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Griffiths, James, Jenkins, Paula, Vargova, Monika et al. (13 more authors) (2018) Enteral lactoferrin to prevent infection for very preterm infants : the ELFIN RCT. Health technology assessment. pp. 1-60. ISSN 2046-4924

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HEALTH TECHNOLOGY ASSESSMENT

VOLUME 22 ISSUE 74 DECEMBER 2018

ISSN 1366-5278

DOI 10.3310/hta22740

Enteral lactoferrin to prevent infection for very preterm infants: the ELFIN RCT

James Griffiths, Paula Jenkins, Monika Vargova, Ursula Bowler, Edmund Juszczak, Andrew King, Louise Linsell, David Murray, Christopher Partlett, Mehali Patel, Janet Berrington, Nicholas Embleton, Jon Dorling, Paul T Heath, William McGuire and Sam Oddie on behalf of the ELFIN Trial Investigators Group

Enteral lactoferrin to prevent infection forvery preterm infants: the ELFIN RCT

James Griffiths,1 Paula Jenkins,1 Monika Vargova,1

Ursula Bowler,1 Edmund Juszczak,1 Andrew King,1

Louise Linsell,1 David Murray,1 Christopher Partlett,1

Mehali Patel,2 Janet Berrington,3 Nicholas Embleton,3

Jon Dorling,4 Paul T Heath,5 William McGuire6*and Sam Oddie7 on behalf of the ELFIN TrialInvestigators Group

1National Perinatal Epidemiology Unit, University of Oxford, Oxford, UK2Bliss, London, UK3Newcastle Neonatal Service, Royal Victoria Infirmary, Newcastle upon Tyne, UK4Queen’s Medical Centre, Nottingham, UK5St George’s, University of London and St George’s University Hospitals NHSTrust, London, UK

6Centre for Reviews and Dissemination, University of York, York, UK7Bradford Institute for Health Research, Bradford, UK

*Corresponding author

Declared competing interests of authors: Since November 2013, Edmund Juszczak has been a member

of the National Institute for Health Research (NIHR) Health Technology Assessment (HTA) General Funding

Committee and HTA Commissioning Funding Committee. William McGuire is a member of the NIHR HTA

Commissioning Board and the NIHR HTA and Efficacy and Mechanism Evaluation Editorial Board. Jon Dorling

is a member of the NIHR HTA General Board (from 2017) and the Maternal, Neonatal and Child Health Panel

(from 2013). Nicholas Embleton reports grants from Prolacta Biosciences Inc. (Duarte, CA, USA), grants from

Danone Nutricia Early Life Nutrition (Paris, France), personal fees from Nestlé Nutrition Institute (Vevey,

Switzerland) and personal fees from Baxter Healthcare Ltd (Newbury, UK) outside the submitted work.

Published December 2018

DOI: 10.3310/hta22740

This report should be referenced as follows:

Griffiths J, Jenkins P, Vargova M, Bowler U, Juszczak E, King A, et al. Enteral lactoferrin to prevent

infection for very preterm infants: the ELFIN RCT. Health Technol Assess 2018;22(74).

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Abstract

Enteral lactoferrin to prevent infection for very preterminfants: the ELFIN RCT

James Griffiths,1 Paula Jenkins,1 Monika Vargova,1 Ursula Bowler,1

Edmund Juszczak,1 Andrew King,1 Louise Linsell,1 David Murray,1

Christopher Partlett,1 Mehali Patel,2 Janet Berrington,3

Nicholas Embleton,3 Jon Dorling,4 Paul T Heath,5 William McGuire6*and Sam Oddie7 on behalf of the ELFIN Trial Investigators Group

1National Perinatal Epidemiology Unit, University of Oxford, Oxford, UK2Bliss, London, UK3Newcastle Neonatal Service, Royal Victoria Infirmary, Newcastle upon Tyne, UK4Queen’s Medical Centre, Nottingham, UK5St George’s, University of London and St George’s University Hospitals NHS Trust, London, UK6Centre for Reviews and Dissemination, University of York, York, UK7Bradford Institute for Health Research, Bradford, UK

*Corresponding author [email protected]

Background: Infections acquired in hospital are an important cause of morbidity and mortality in very

preterm infants. Several small trials have suggested that supplementing the enteral diet of very preterm

infants with lactoferrin, an antimicrobial protein processed from cow’s milk, prevents infections and

associated complications.

Objective: To determine whether or not enteral supplementation with bovine lactoferrin (The Tatua

Cooperative Dairy Company Ltd, Morrinsville, New Zealand) reduces the risk of late-onset infection

(acquired > 72 hours after birth) and other morbidity and mortality in very preterm infants.

Design: Randomised, placebo-controlled, parallel-group trial. Randomisation was via a web-based portal

and used an algorithm that minimised for recruitment site, weeks of gestation, sex and single versus

multiple births.

Setting: UK neonatal units between May 2014 and September 2017.

Participants: Infants born at < 32 weeks’ gestation and aged < 72 hours at trial enrolment.

Interventions: Eligible infants were allocated individually (1 : 1 ratio) to receive enteral bovine lactoferrin

(150 mg/kg/day; maximum 300 mg/day) or sucrose (British Sugar, Peterborough, UK) placebo (same dose)

once daily from trial entry until a postmenstrual age of 34 weeks. Parents, caregivers and outcome

assessors were unaware of group assignment.

Outcomes: Primary outcome – microbiologically confirmed or clinically suspected late-onset infection.

Secondary outcomes – microbiologically confirmed infection; all-cause mortality; severe necrotising

enterocolitis (NEC); retinopathy of prematurity (ROP); bronchopulmonary dysplasia (BPD); a composite of

infection, NEC, ROP, BPD and mortality; days of receipt of antimicrobials until 34 weeks’ postmenstrual age;

length of stay in hospital; and length of stay in intensive care, high-dependency and special-care settings.

DOI: 10.3310/hta22740 HEALTH TECHNOLOGY ASSESSMENT 2018 VOL. 22 NO. 74

© Queen’s Printer and Controller of HMSO 2018. This work was produced by Griffiths et al. under the terms of a commissioning contract issued by the Secretary of State forHealth and Social Care. This issue may be freely reproduced for the purposes of private research and study and extracts (or indeed, the full report) may be included in professionaljournals provided that suitable acknowledgement is made and the reproduction is not associated with any form of advertising. Applications for commercial reproduction shouldbe addressed to: NIHR Journals Library, National Institute for Health Research, Evaluation, Trials and Studies Coordinating Centre, Alpha House, University of Southampton SciencePark, Southampton SO16 7NS, UK.

v

Results: Of 2203 enrolled infants, primary outcome data were available for 2182 infants (99%). In the

intervention group, 316 out of 1093 (28.9%) infants acquired a late-onset infection versus 334 out of 1089

(30.7%) infants in the control group [adjusted risk ratio (RR) 0.95, 95% confidence interval (CI) 0.86 to 1.04].

There were no significant differences in any secondary outcomes: microbiologically confirmed infection

(RR 1.05, 99% CI 0.87 to 1.26), mortality (RR 1.05, 99% CI 0.66 to 1.68), NEC (RR 1.13, 99% CI 0.68 to

1.89), ROP (RR 0.89, 99% CI 0.62 to 1.28), BPD (RR 1.01, 99% CI 0.90 to 1.13), or a composite of infection,

NEC, ROP, BPD and mortality (RR 1.01, 99% CI 0.94 to 1.08). There were no differences in the number of

days of receipt of antimicrobials, length of stay in hospital, or length of stay in intensive care, high-dependency

or special-care settings. There were 16 reports of serious adverse events for infants in the lactoferrin group

and 10 for infants in the sucrose group.

Conclusions: Enteral supplementation with bovine lactoferrin does not reduce the incidence of infection,

mortality or other morbidity in very preterm infants.

Future work: Increase the precision of the estimates of effect on rarer secondary outcomes by combining

the data in a meta-analysis with data from other trials. A mechanistic study is being conducted in a

subgroup of trial participants to explore whether or not lactoferrin supplementation affects the intestinal

microbiome and metabolite profile of very preterm infants.

Trial registration: Current Controlled Trials ISRCTN88261002.

Funding: This project was funded by the National Institute for Health Research (NIHR) Health Technology

Assessment programme and will be published in full in Health Technology Assessment; Vol. 22, No. 74.

See the NIHR Journals Library website for further project information. This trial was also sponsored by the

University of Oxford, Oxford, UK. The funder provided advice and support and monitored study progress

but did not have a role in study design or data collection, analysis and interpretation.

ABSTRACT


vi

Contents

List of tables ix

List of figures xi

List of boxes xiii

List of abbreviations xv

Plain English summary xvii

Scientific summary xix

Chapter 1 Introduction 1

Late-onset infection in very preterm infants 1

Diagnosis, treatment and prevention of late-onset invasive infection 1

Lactoferrin 2

Bovine lactoferrin 2

Lactoferrin supplementation 3

Existing evidence 3

Objective 3

Chapter 2 Methods 5

Design 5

Ethics approval and research governance 5

Patient and public involvement 5

Participants 5

Inclusion criteria 5

Exclusion criteria 5

Informed consent and recruitment 6

Intervention 7

Investigational Medicinal Product management 7

Investigational Medicinal Product prescription and preparation 7

Randomisation 8

Allocation concealment and blinding 8

Stopping Investigational Medicinal Product 8

Masking 8

Internal pilot 8

Outcomes 9

Primary outcome 9

Secondary outcomes 9

Sample size 10

Statistical analyses 11

Data collection 11

Adverse event reporting 11

Suspected unexpected serious adverse reactions 12

Development safety update report 12



vii

Economic analysis (planned) 12

Governance and monitoring 12

Summary of changes to the study protocol 13

Chapter 3 Results 15

Recruitment and retention 15

Demographic and other baseline characteristics 15

Adherence 17

Outcomes 17

Primary outcome 17


Economic analysis 20

Safety and adverse events 20

Post hoc analyses 22

Chapter 4 Discussion and conclusions 25

Summary of main findings 25

Limitations 26


Cost analyses 27

Qualitative analysis and parent views 27

Long-term outcomes 27

Applicability 27

Implications for practice 28

Implications for research 28

Acknowledgements 29

References 31

Appendix 1 Recruiting neonatal units 37

Appendix 2 Continuing care sites 39

Appendix 3 Preparation of Investigational Medicinal Product for administration 41

Appendix 4 Case definition of necrotising enterocolitis 43

Appendix 5 British Association of Perinatal Medicine: ‘categories of care’ 45

Appendix 6 Data collection forms 47

Appendix 7 Safety reporting: definitions 49

Appendix 8 Summary of changes to the study protocol 51

Appendix 9 Withdrawals from intervention by randomisation group 57

Appendix 10 Group allocation per recruiting site 59

CONTENTS


viii

List of tables

TABLE 1 Participants required per arm by CER 10

TABLE 2 Infant and maternal characteristics at randomisation 16

TABLE 3 Adherences measures 18

TABLE 4 Primary and secondary outcomes 18

TABLE 5 Subgroup analyses for confirmed or suspected late-onset infection 20

TABLE 6 List of SAEs reported by randomisation group 21

TABLE 7 Microbiologically confirmed late-onset infection by classification of

micro-organism 23

TABLE 8 Number of episodes of confirmed or suspected sepsis 24

TABLE 9 Late-onset infection from trial entry until hospital discharge by

exposure to probiotics 24



ix

List of figures

FIGURE 1 Flow of participants through the trial 15

FIGURE 2 Subgroup analyses for confirmed or suspected late-onset invasive

infection 21



xi

List of boxes

BOX 1 Lactoferrin: mechanisms of action 2

BOX 2 Summary findings of the Cochrane review meta-analyses 3

BOX 3 Definition of microbiologically confirmed late-onset infection 9

BOX 4 Definition of clinically suspected late-onset infection 10

BOX 5 Classification of micro-organisms 23



xiii

List of abbreviations

ARR absolute risk reduction

BPD bronchopulmonary dysplasia

CER control event rate

CI confidence interval

CPAP continuous positive airways

pressure

CRF case report form

CSF cerebrospinal fluid

DMC Data Monitoring Committee

ELFIN Enteral Lactoferrin In Neonates

GMP good manufacturing practice

ID identification

IMP Investigational Medicinal Product

IQR interquartile range

MHRA Medicines and Healthcare products

Regulatory Agency

NEC necrotising enterocolitis

NIHR National Institute for Health

Research

NPEU CTU National Perinatal Epidemiology

Unit Clinical Trials Unit

PI principal investigator

PIL parent information leaflet

RCT randomised controlled trial

REC Research Ethics Committee

ROP retinopathy of prematurity

RR risk ratio

SAE serious adverse event

SAR serious adverse reaction

SD standard deviation

SIFT Speed of Increasing Milk Feeds Trial

SOP standard operating procedure

SUSAR suspected unexpected serious

adverse reaction

TSC Trial Steering Committee



xv

Plain English summary

Babies who are born ‘very prematurely’ (i.e. > 8 weeks early) need specialist hospital care on a neonatal

unit. These babies can develop serious infections and illnesses during their stay in hospital. The risk

of developing such infections is highest in the most premature infants.

This study was designed to test whether or not giving lactoferrin (The Tatua Cooperative Dairy Company Ltd,

Morrinsville, New Zealand), a naturally occurring milk protein (often used as a food supplement), to babies

can help to protect them against infections. A small study was previously carried out in Italy and, although

the results were promising, we needed to find out more. A large study was undertaken in neonatal units

across the UK. More than 2200 very premature babies took part to find out whether or not lactoferrin is

effective at preventing infections and other illnesses. With consent from babies’ parents, clinicians randomly

(by chance) allocated babies to receive either lactoferrin or sugar (sham treatment) mixed with milk once a

day until they were no longer at a high risk of serious infections (the equivalent of 34 weeks’ gestation).

The babies’ parents, nurses and doctors were not aware of whether each individual baby was receiving

lactoferrin or sucrose (British Sugar, Peterborough, UK).

When all of the study data had been analysed, it was found that supplemental lactoferrin did not reduce

the risk of infection or other serious illness or death, or affect the length of hospital stay, in very premature

babies born > 8 weeks early. Because the study was large and used reliable methods, these results prove

that lactoferrin supplementation does not have important benefits for very premature babies and that

there is no need for any further research into the use of this treatment.



xvii


Scientific summary

Background

Late-onset infection is the most common serious complication associated with hospital care for preterm infants. The reported incidence ranges from about 15% to 30% in very preterm infants, reflecting their high levels of exposure to invasive procedures and intensive care. Very preterm infants with late-onset infection have a higher rate of mortality and morbidities, such as bronchopulmonary dysplasia (BPD), necrotising enterocolitis (NEC) and retinopathy of prematurity (ROP), than infants without infections. Given this burden of mortality, morbidity and the associated costs for families and health services, the James Lind Alliance Preterm Birth Priority Setting Partnership (Southampton, UK) has identified the development and assessment of better methods to prevent infection in preterm infants as a research priority. One such promising intervention is enteral supplementation with the processed cow’s milk protein, lactoferrin (The Tatua Cooperative Dairy Company Ltd, Morrinsville, New Zealand).

Lactoferrin is the major whey protein in breast milk and is a key component of the mammalian innate response to infection. It has broad microbiocidal activity via mechanisms, such as cell membrane disruption, iron sequestration, the inhibition of microbial adhesion to host cells and the prevention of biofilm formation. Lactoferrin has prebiotic properties, promoting the growth of beneficial bacteria and reducing colonisation by pathogenic species. It enhances intestinal mucosal integrity and immune function by modulating cytokine expression, suppressing free-radical activity and activating and mobilising leucocytes.

Bovine lactoferrin is 70% homologous with human lactoferrin, but has a higher antimicrobial activity. It has been a component of the human infant diet for thousands of years, is available as a food supplement in a powder form and is registered as ‘Generally Recognised as Safe’ by the US Federal Drug Administration.

The Cochrane review of lactoferrin supplementation for preterm infants includes six randomised controlled trials (RCTs) with 1071 participants in total (Pammi M, Suresh G. Enteral lactoferrin supplementation for prevention of sepsis and necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev 2017;6: CD007137). Meta-analyses suggest that lactoferrin reduces the incidence of late-onset invasive infection by about 40%. The effect size is similar whether infants are fed human milk or formula. The risk of NEC is decreased by about 60%. No evidence of adverse effects or intolerance exists. As the included trials were small and contained various methodological weaknesses, the evidence was considered to be of low quality and the review concluded that data from high-quality trials were needed to provide evidence of sufficient validity to inform policy and practice.

Objective

To assess the effect of enteral supplementation with bovine lactoferrin on the risk of late-onset infection and other morbidity and mortality in very preterm infants.

Methods

Study designThe Enteral Lactoferrin In Neonates (ELFIN) trial: a multicentre, randomised, placebo-controlled, parallel-group trial of prophylactic enteral lactoferrin supplementation to prevent late-onset invasive infection in very preterm infants (see www.npeu.ox.ac.uk/elfin).

© Queen’s Printer and Controller of HMSO 2018. This work was produced by Griffiths et al. under the terms of a commissioning contract issued by the Secretary of State for Health and Social Care. This issue may be freely reproduced for the purposes of private research and study and extracts (or indeed, the full report) may be included in professional journals provided that suitable acknowledgement is made and the reproduction is not associated with any form of advertising. Applications for commercial reproduction should be addressed to: NIHR Journals Library, National Institute for Health Research, Evaluation, Trials and Studies Coordinating Centre, Alpha House, University of Southampton Science Park, Southampton SO16 7NS, UK.

xix

https://www.npeu.ox.ac.uk/elfin

SettingNeonatal units in UK hospitals; participant recruitment and initial care in 37 units and continuing care during

birth hospitalisation in a further 97 units.

ParticipantsVery preterm infants of < 72 hours old at randomisation. Infants with a severe congenital anomaly, without

a realistic prospect of survival or who were likely to be fasted enterally for > 14 days were ineligible to

participate. Written consent was sought from parents only after they had received a verbal and written

explanation of the trial.

InterventionsInfants were allocated individually via a secure web-based randomisation portal in the ratio of 1 : 1, minimised

for recruiting site, sex, gestational age at birth (completed weeks) and single versus multiple births, to receive

either (1) bovine lactoferrin (150mg/kg/day, up to a maximum of 300 mg/day) or (2) sucrose (British Sugar,

Peterborough, UK) placebo (at the same dose). The lactoferrin powder or sucrose was mixed in sterile water

plus either expressed breast milk or formula prior to administration via a nasogastric or orogastric tube or

orally. The intervention was commenced when the infant’s enteral feed volume reached 12ml/kg/day and

was continued once daily until the infant reached 34 weeks’ postmenstrual age. Parents, clinicians, carers and

those assessing the outcomes were unaware of group assignment.

Outcomes

Primary outcomeMicrobiologically confirmed or clinically suspected late-onset infection (occurring > 72 hours after birth)

from trial entry until hospital discharge.

Secondary outcomesMicrobiologically confirmed infection; all-cause mortality; severe NEC (Bell’s stage II or III); ROP treated

surgically or medically; BPD (receipt of supplemental oxygen or respiratory support at 36 weeks’

postmenstrual age); a composite of infection, NEC, ROP, BPD and mortality; days of administration of

antimicrobials until 34 weeks’ postmenstrual age; duration of birth hospitalisation; and length of stay in

intensive care, high-dependency care or special-care settings.

Statistics and analysis plan

Sample sizeCalculations were based on a primary outcome event rate range of 18% to 24%. In summary, with 90%

power and a two-sided 5% significance level, to detect an absolute risk reduction of 5–5.8% (relative risk

reduction of 24–28%) required a trial with a total of up to 2200 participants (allowing for an anticipated

loss to follow-up of up to 5%). This sample size was sufficient to exclude important effects on secondary

outcomes with 90% power.

Statistical analysesDemographic factors and clinical characteristics at randomisation were summarised with counts (%) for

categorical variables, mean (standard deviation) for normally distributed continuous variables or median

[interquartile range (IQR)] for other continuous variables.

Outcomes for participants were analysed in the groups to which they were assigned, regardless of deviation

from the protocol or treatment received. Comparative analyses calculated the relative risk ratio (RR) with

95% confidence interval (CI) for the primary outcome (99% CIs for all other dichotomous outcomes), the

mean difference (99% CI) for normally distributed continuous outcomes or the median difference (99% CI)

for skewed continuous variables.

SCIENTIFIC SUMMARY


xx

The groups were compared using regression analysis adjusting for the minimisation factors (recruiting hospital,

sex, weeks’ gestation at birth and single vs. multiple births) to account for the correlation between treatment

groups introduced by balancing the randomisation. The crude unadjusted and adjusted estimates were

calculated with the primary inference to be based on the adjusted analysis.

The consistency of the effect of lactoferrin supplementation on the primary outcome across specific subgroups

of infants was assessed using the statistical test of interaction. Prespecified subgroups were (1) completed

weeks’ gestation at birth and (2) infants given human breast milk versus formula versus both human milk and

formula during the trial period.

Results

The internal pilot was undertaken from May 2014 for 12 months in six neonatal units; 90 infants were

recruited to participate. The main trial recruitment period ran from July 2015 to September 2017 in

37 neonatal units. In total, the trial recruited 2203 infants; 1099 infants were allocated to receive bovine

lactoferrin and 1104 were allocated to receive sucrose placebo. Four infants had consent withdrawn or

unconfirmed. In total, 1098 infants in the lactoferrin group and 1101 in the sucrose group were included

in the intention-to-treat analyses.

Baseline characteristics were well balanced. The median gestation age at birth was 29 weeks in both groups

(37% aged < 28 weeks). The median birthweight was 1126 g in the lactoferrin group and 1143 g in the

placebo group. Overall, 91% of infants were exposed to antenatal corticosteroid, 57% were born via

caesarean section, 25% were born following rupture of maternal amniotic membranes for > 24 hours and

12% had evidence of absent or reverse end diastolic flow in the fetal umbilical artery.

Primary outcomeData were available for 2182 infants (99%). In the intervention group, 316 out of 1093 (28.9%) infants

acquired a microbiologically confirmed or clinically suspected late-onset infection, compared with 334 out

of 1089 (30.7) in the control group (adjusted RR 0.95, 95% CI 0.86 to 1.04).

Secondary outcomesThere were no significant differences in rates of:

l microbiologically confirmed infection: RR 1.05 (99% CI 0.87 to 1.26)l all-cause mortality until hospital discharge: RR 1.05 (99% CI 0.66 to 1.68)l NEC: RR 1.13 (99% CI 0.68 to 1.89)l ROP: RR 0.89 (99% CI 0.62 to 1.28)l BPD: RR 1.01 (99% CI 0.90 to 1.13)l a composite of infection, NEC, ROP, BPD and mortality: RR 1.01 (99% CI 0.94 to 1.08).

There were no differences in the median number of days of receipt of:

l antimicrobials: median difference 0 (99% CI –1 to 1)l hospital care: median difference 1 (99% CI –1 to 3)l intensive care: median difference 0 (99% CI –1 to 1)l high-dependency care: median difference 1 (99% CI –1 to 3)l special care: median difference –1 (99% CI –3 to 1).

Subgroup analyses did not show any significant interactions for:

l completed weeks’ gestation at birth: p = 0.273l type of enteral milk received (human, formula, or both): p = 0.400.



xxi

Safety and adverse eventsThere were 16 serious adverse events (SAEs) reported for infants in the lactoferrin group (six severe) and

10 for infants in the sucrose group (three severe). Two SAEs, both in the lactoferrin group, were assessed

as being ‘possibly related’ to the trial intervention. The remaining 24 SAEs were considered to be unrelated

to the trial intervention.

Discussion

The ELFIN trial shows that enteral supplementation of bovine lactoferrin (150 mg/kg/day until 34 weeks’

postmenstrual age) does not reduce the risk of late-onset infection, other morbidity or mortality in very

preterm infants. This contradicts the existing trial evidence. The current Cochrane review includes six RCTs

and meta-analyses that suggest substantial reductions in the risk of late-onset infection and NEC associated

with lactoferrin supplementation in very preterm infants. The included trials were small and some contained

other design and methodological weaknesses that may have introduced biases, thus resulting in overestimation

of the effect sizes. The largest previous trial, in which 331 infants participated, showed a relative risk reduction

of 66% for late-onset infection. This effect size estimate may have been inflated by performance and detection

bias, and by methodological weaknesses, including the absence of predefined criteria for interim analyses.

Given these concerns, the Cochrane review graded the quality of the existing evidence for effects on key

outcomes as ‘low’ and concluded that data from methodologically rigorous RCTs were needed to generate

evidence of sufficient validity to inform policy and practice.

The ELFIN trial provides these data. The validity of the findings is enhanced by the methodological quality

and power of the trial. Best practices were used to limit bias, including central web-based randomisation

for allocation concealment, blinding of parents, caregivers and investigators to the group allocation,

and intention-to-treat analyses of outcomes based on a prespecified statistical analysis plan. The trial

recruited > 2200 participants as per protocol and a priori sample size estimation. Demographic and

prognostic characteristics were well-balanced between the two groups at randomisation with a minimisation

algorithm, ensuring balance for known or putative prognostic indicators (completed weeks’ gestation, sex,

single vs. multiple births) or potential confounding influences (recruiting site). Interim analyses by the trial’s

independent Data Monitoring Committee (DMC) used criteria to minimise the chances of spurious findings

caused by data fluctuations before a sufficient sample size was achieved. Adherence to the allocated

interventions was high, the incidence of protocol violations was low and outcome data were available for

> 99% of the trial cohort. Event rates for the primary and secondary outcomes were similar to those that

were anticipated and as described in other cohort studies and RCTs involving very preterm infants.

The trial had sufficient power to detect important effects on the risk of late-onset infection and other

morbidities. More precise estimates of effect were able to be generated than were available previously.

The 95% CI for the relative risk estimate for the primary outcome excludes a > 14% risk reduction and

a ≥ 4% increase in risk. These estimates were consistent across gestational ages at birth and were not

affected by the type of enteral feeds that infants received during the trial period (human milk, formula or

both). It is therefore unlikely that lactoferrin has any important benefits for subgroups of infants at higher

risk of infection.

Estimates for the secondary outcomes indicated consistently that lactoferrin supplementation does not have

important effects on the risk of major morbidities. An analysis was prespecified of the effect on a composite

of infection, NEC, BPD, ROP and mortality. The adjusted RR point estimate for this secondary outcome

was 1.01, with a 99% CI excluding a > 6% reduction and a ≥ 8% increase in risk. As these morbidities,

particularly infection and NEC, are the major reasons for the receipt of invasive interventions and higher levels

of care in very preterm infants, it is not surprising that there were no effects shown on the level of exposure to

antimicrobial agents, or on the duration of hospitalisation or stay in intensive or high-dependency care settings.

SCIENTIFIC SUMMARY


xxii

Given that the ELFIN trial did not show any differences between groups in the risk of morbidity or on levels

of care received, we do not plan to apply for permission and further funding to assess longer-term outcomes

of trial participants. Any between-group differences in growth and neurodevelopmental outcomes are

predicated largely on differences in the incidence of late-onset infections, NEC and associated morbidities.

As these differences were not shown, there is no longer an impelling rationale for expecting lactoferrin

supplementation to have an impact on long-term growth or development.

The ELFIN trial findings are applicable in the UK and internationally. Participants were enrolled in 37 neonatal

units across the country, providing broad geographical, and social and ethnic representation. Many infants

who were recruited were transferred subsequently to another neonatal unit, typically closer to the family

home, for ongoing care. Trial participation continued in these additional 97 neonatal units and this practice

mirrors managed clinical network care pathways for very preterm infants in the UK. The trial population was

representative of very preterm infants cared for within health-care facilities in well-resourced health services

and included a substantial proportion of extremely preterm infants (37%) and of infants with other putative

risk factors for neonatal morbidity, such as prolonged rupture of maternal amniotic membranes (25%) and

evidence of absent or reverse end diastolic flow in the fetal umbilical artery (12%). Overall, about 30% of

participants acquired a microbiologically confirmed or clinically suspected late-onset infection, and about

17% in total had a microbiologically confirmed infection, consistent with rates reported from cohort studies

and other RCTs. Similarly, the incidence of NEC (about 5%) was similar to rates reported in large,

population-based surveillance studies and RCTs.

Conclusion

These findings do not support the use of enteral bovine lactoferrin supplementation to prevent late-onset

infection or other morbidity in very preterm infants. Research efforts could continue to investigate the

aetiology, epidemiology and pathogenesis of late-onset infection and related morbidities, and to develop,

refine and assess other interventions that may prevent or reduce adverse acute and long-term consequences

for very preterm infants and their families.

Trial registration

This trial is registered as ISRCTN88261002.

Funding

Funding for this study was provided by the Health Technology Assessment programme of the National

Institute for Health Research. This trial was also sponsored by the University of Oxford, Oxford, UK. The

funder provided advice and support and monitored study progress but did not have a role in study design

or data collection, analysis and interpretation.



xxiii

Chapter 1 Introduction

Late-onset infection in very preterm infants

Late-onset invasive infection (occurring > 72 hours after birth) is the most common serious complication

associated with hospital care for preterm infants. The UK James Lind Alliance Preterm Birth Priority Setting

Partnership has identified the development and assessment of better methods to prevent infection in

preterm infants as a research priority.1

The incidence of late-onset infection is typically estimated to be > 20% in very preterm infants, reflecting the

level and duration of exposure to invasive procedures and intensive care.2,3 Very preterm infants who acquire a

late-onset bloodstream or deep-seated infection are at higher risk of mortality and a range of acute morbidities

including necrotising enterocolitis (NEC), retinopathy of prematurity (ROP) and bronchopulmonary dysplasia

(BPD) than comparable infants without infection.4–6 Over the long term, late-onset infection is associated with

higher rates of adverse neurodevelopmental outcomes, including visual, hearing and cognitive impairment, and

cerebral palsy.5

Mortality and morbidity are usually associated with Gram-negative bacterial, Staphylococcus aureus or fungal

bloodstream infections or meningitis.7–9 Coagulase-negative staphylococcal infection, despite accounting for

about half of all infections, is generally associated with a more benign clinical course.10 Meningitis and other

deep-seated infections are rare and the mortality rate is lower than that attributed to Gram-negative or other

Gram-positive bacterial infections. However, even low-grade coagulase-negative staphylococcal bloodstream

infections may generate inflammatory cascades that are associated with both acute morbidity (metabolic,

respiratory or thermal instability, thrombocytopenia) and long-term white matter and other brain damage

that may result in adverse neurodevelopmental outcomes.11

As a consequence of associated morbidities, very preterm infants with late-onset infection may spend about

20 more days in hospital than gestation-comparable infants without infection.12 Late-onset infection and

associated morbidities therefore have major consequences for perinatal health-care service management,

delivery and costs.

Diagnosis, treatment and prevention of late-onset invasive infectionClinical signs and laboratory markers may be unreliable predictors of true late-onset infection, especially in

very preterm infants.13,14 A policy of early empirical treatment of suspected infection is usually implemented.

Most neonates who are treated as a result of ‘sepsis evaluation’, however, do not have infection confirmed

subsequently.15 This results in unnecessary exposure to antibiotics, which not only subjects very preterm

infants to more interventions but may drive the emergence of antibiotic-resistant pathogens in the neonatal

unit.16,17 Although generic infection control measures, such as hand-washing and intravascular catheter

‘care bundles’, have helped to prevent some episodes of late-onset invasive infection in very preterm infants,

benchmarking and quality improvement studies in neonatal networks have indicated that there is a need for

measures to further reduce the incidence.18

Given this burden of mortality, acute and long-term morbidity, and costs to families and health services,

there is a need to develop innovative strategies to prevent late-onset invasive infection in very preterm

infants.19



1

Lactoferrin

Lactoferrin, a member of the transferrin family of iron-binding glycoproteins, is a key component of the

mammalian innate response to infection.20–22 It is the major whey protein in human colostrum, present at a

concentration of about 6 mg/ml and is present in mature breast milk at a concentration of about 1 mg/ml.23

Lactoferrin is also present in mammalian tears, saliva, cerebrospinal fluid (CSF) and other secretions.22

Lactoferrin has broad microbiocidal activity by mechanisms, such as cell membrane disruption, iron

sequestration, the inhibition of microbial adhesion to host cells and the prevention of biofilm formation

(Box 1).20,21 Development of resistance to lactoferrin is improbable as it would require multiple simultaneous

mutations. Lactoferrin remains a potent inhibitor of viruses, bacteria, fungi and protozoa after millions of

years of mammalian evolution.24,25

Lactoferrin has prebiotic properties, creating an enteric environment that promotes the growth of beneficial

bacteria and reducing colonisation with pathogenic species.26,27 It has direct intestinal immunomodulatory

and anti-inflammatory actions mediated by modulating cytokine expression, mobilising leucocytes into the

circulation and activating T-lymphocytes.28,29 At high concentrations, as in colostrum, lactoferrin enhances

the proliferation of enterocytes and the closure of enteric gap junctions.30 At lower concentrations,

lactoferrin stimulates the differentiation of enterocytes and the expression of intestinal digestive enzymes.31

Lactoferrin suppresses free-radical activity when iron is added to milk, suggesting that it may have further

anti-inflammatory actions that could modulate the pathogenesis of diseases linked with free-radical

generation, such as NEC, ROP and BPD.32

Bovine lactoferrinBovine lactoferrin (The Tatua Cooperative Dairy Company Ltd, Morrinsville, New Zealand) is > 70%

homologous with human lactoferrin but has higher antimicrobial activity. It is inexpensive compared with

BOX 1 Lactoferrin: mechanisms of action

l Antimicrobial effects:

¢ cell membrane disruption

¢ iron sequestration

¢ inhibition of microbial adhesion to host cells

¢ prevention of biofilm formation.

l Prebiotic effects:

¢ promote intestinal growth of beneficial bacteria (probiotics)

¢ reduce colonisation with pathogenic species.

l Immune-modulatory and anti-inflammatory actions:

¢ modulate cytokine expression

¢ mobilise leucocytes into the circulation

¢ activate T-lymphocytes

¢ suppress free-radical activity.

l Intestinal integrity effects:

¢ stimulate differentiation and proliferation of enterocytes

¢ promote closure of enteric gap junctions

¢ increase expression of intestinal digestive enzymes.

INTRODUCTION


2

human or recombinant lactoferrin and is available commercially as a food supplement in a stable powder

form.33 The affinity of bovine lactoferrin for the human small intestine lactoferrin receptor is low and intact

lactoferrin and digested fragments (lactoferricins), which also have high microbiocidal activity, are excreted

enterally.34,35 Bovine lactoferrin has been a component of the human infant diet for thousands of years and

is registered as ‘Generally Recognised As Safe’ by the US Federal Drug Administration with no reports of

human toxicity.36 The ‘no observed adverse effect level’ is > 2 g/kg/day in rodents.37 Given the absence of

adverse effects, the European Food Safety Authority Panel concluded that bovine lactoferrin for infants

is safe at the standard supplementation levels (up to about 210 mg/kg of body weight per day).38

Lactoferrin supplementation

Very preterm infants typically ingest little or no milk during the early neonatal period and thus have low

lactoferrin intake. This deficiency may be further exacerbated by delays in establishing enteral feeding.

Enteral lactoferrin supplementation has been proposed and assessed as a simple strategy to compensate

for this gestational immunodeficiency.39

Existing evidenceThe Cochrane review by Pammi and Suresh40 identified six completed randomised controlled trials (RCTs),

involving 1071 participants in total.41–46 Meta-analyses suggest that enteral supplementation with lactoferrin

reduces the incidence of late-onset invasive infection by about 40%; the effect size is similar whether

infants are fed predominantly human milk or formula milk. The incidence of NEC is decreased by about

60%. No evidence of an effect on all-cause mortality was found and no adverse effects or intolerance were

reported (Box 2). However, because the trials were generally small and contained various methodological

weaknesses that increased the risk of selection and performance bias, and because meta-analyses were

limited by data availability and heterogeneity, the Grading of Recommendations Assessment, Development

and Evaluation (GRADE) working group assessment of the quality of this evidence was ‘low’, meaning that

further research was likely to have an important impact on the confidence in the estimates of effect and is

likely to change these estimates. The Cochrane review concluded that additional data from large, good-quality

RCTs of lactoferrin supplementation in very preterm infants were needed to enhance the validity and

applicability of the evidence-base sufficiently to inform policy and practice.40

Objective

The study aimed to assess the effect of enteral administration of bovine lactoferrin on the incidence of

late-onset infection, other morbidity and mortality in very preterm infants.

BOX 2 Summary findings of the Cochrane review meta-analyses40

l Late-onset infection: typical RR 0.59, 95% CI 0.40 to 0.87; typical RD –0.06, 95% CI –0.10 to –0.02;

number needed to treat for an additional beneficial outcome was 17, 95% CI 10 to 50; six trials,

886 participants.

l Necrotising enterocolitis (Bell’s stage II or III): typical RR 0.40, 95% CI 0.18 to 0.86; typical RD –0.04, 95% CI

–0.06 to –0.01; number needed to treat for an additional beneficial outcome was 25, 95% CI 17 to 100;

four trials, 750 participants.

l All-cause mortality: typical RR 0.65, 95% CI 0.37 to 1.11; typical RD –0.02, 95% CI –0.05 to 0; six trials,

1071 participants.

CI, confidence interval; RD, risk difference; RR, risk ratio.



3

Chapter 2 Methods

Design

The Enteral Lactoferrin In Neonates (ELFIN) trial was a UK, multicentre, parallel-group, placebo-controlled

RCT (see www.npeu.ox.ac.uk/elfin).47

Ethics approval and research governance

The ELFIN trial protocol was approved by the National Research Ethics Service Committee East Midlands –

Nottingham 2 on 2 April 2013 (reference number 13/EM/0118).

Local approval and site-specific assessments were obtained from the NHS trusts for trial sites.

The trial was registered with the International Standard Randomised Controlled Trial Register

(https://doi.org/10.1186/ISRCTN88261002).

Patient and public involvement

During the development and delivery of the ELFIN trial, we engaged with infant and family representatives

experienced in voicing service users’ views (principally via Bliss, a UK national charity supporting preterm or

sick newborn infants and their families: www.bliss.org.uk/). Parents with children who had received neonatal

intensive care contributed via Bliss and directly to the development of trial materials (e.g. parent information

and resources) and research staff training (e.g. in simulated sessions on ‘seeking consent’). We adhered

to INVOLVE good practice guidelines to ensure service-user leadership in the delivery of the trial and

dissemination of the findings (www.invo.org.uk/).

Participants

Inclusion criteria

l Gestational age at birth of < 32 weeks.l < 72 hours old at randomisation.l Written informed parental consent.

Exclusion criteria

l Severe congenital anomaly.l Anticipated enteral fasting for > 14 days.l No realistic prospect of survival.

Infants receiving antibiotic treatment at randomisation were eligible to participate.



5

https://www.npeu.ox.ac.uk/elfin

https://doi.org/10.1186/ISRCTN88261002

https://www.bliss.org.uk/

https://www.invo.org.uk/

SettingNeonatal units in the UK caring for very preterm infants:

l recruiting sites where parents’ consent was obtained and infants could be recruited and randomised to

commence participation in the trial (n = 37; see Appendix 1)l continuing care sites where clinicians continued to administer the intervention and collect routine data

if a participating infant of < 34 weeks’ postmenstrual age was transferred from a recruiting site

(n = 97; see Appendix 2).

Depending on the interventions being given, it was possible for an infant to participate in other clinical trials at

the same time as participating in the ELFIN trial. The Speed of Increasing Milk Feeds Trial (SIFT) was designed

to allow infants to be enrolled in both trials.48 The ELFIN trial and SIFT shared procedures and, in some cases,

joint data collection forms and other documentation. Other trials being run simultaneously in any units were

discussed by the chief investigators or their delegated representative to agree whether or not joint recruitment

was appropriate and likely to be acceptable.

Screening and eligibility assessmentPotential participants meeting the eligibility criteria were identified by the local health-care team. As the

eligibility criteria did not require specific medical assessment, assessment of eligibility was accepted to be

within the scope of competency of appropriately trained and experienced neonatal nurses, if so delegated

by the principal investigator (PI).

Informed consent and recruitment

Consent was sought from parents of potential participants only after they had received a full verbal

and written explanation of the trial [parent information leaflet (PIL); see www.npeu.ox.ac.uk/elfin/

parent-resources (accessed 29 June 2018)]. Parents who did not speak English were approached only

if an adult interpreter was available.

Informing potential participants’ parents of possible benefits and risks occurred as a staged process.49

If it was likely that the expected infant was eligible to participate in the trial, the PIL and preliminary verbal

information was offered prior to birth. Further verbal information was provided after birth as it was to the

parents of infants who were not identified antenatally.

Written informed parental consent was obtained by means of dated parental signature and the signature

of the person who obtained informed consent: the PI or health-care professional with delegated authority

(see www.npeu.ox.ac.uk/elfin/neonatal-staff). A copy of the signed informed consent form was given

to the parents. A copy was retained in the infant’s medical notes, a copy was retained by the PI and the

original was sent to the Clinical Trials Unit.

Participants or parents were not given any financial or material incentive or compensation to take part. It

was made clear that parents remained free to withdraw their infant from the trial at any time without the

need to provide any reason or explanation. Parents were aware that this decision would have no impact

on any aspects of their infant’s continuing care.

The trial entry form was completed after informed consent had been given. The recorded information

was entered on to the National Perinatal Epidemiology Unit Clinical Trials Unit (NPEU CTU) randomisation

website [see https://rct.npeu.ox.ac.uk/ (accessed 29 June 2018)]. Infants were considered to have been

enrolled once they have been given a study number and have been allocated a treatment pack number by

the randomisation facility.

METHODS


6

https://www.npeu.ox.ac.uk/elfin/parent-resources

https://www.npeu.ox.ac.uk/elfin/parent-resources

https://www.npeu.ox.ac.uk/elfin/neonatal-staff

https://rct.npeu.ox.ac.uk/

Intervention

Trial participants were allocated randomly to receive either:

l bovine lactoferrin orl sucrose (British Sugar, Peterborough, UK).

The UK Medicines and Healthcare Regulatory Agency (MHRA) indicated that, for the purposes of the trial,

the intervention and sucrose placebo were considered to be Investigational Medicinal Products (IMPs)

and subject to good manufacturing practice (GMP) regulations. After discussion with MHRA, the IMP

was considered to be ‘category B’ (risk slightly above routine practice because bovine lactoferrin is not a

licensed product).

Bulk lactoferrin was imported from The Tatua Cooperative Dairy Company Ltd, a New Zealand-based

company that manufactures highly purified powder (see www.tatua.com/specialty-nutritionals-ingredients/

lactoferrin/).

Bulk sucrose was obtained in the UK from British Sugar (see www.britishsugar.co.uk/).

The IMP was packaged into individual doses in sealed opaque containers and assembled into participant

packs to GMP in the MHRA-approved NHS clinical trials pharmacy unit at the Royal Victoria Hospital,

Newcastle upon Tyne (www.newcastle-hospitals.org.uk/services/Pharmacy_services_newcastle-specials-

pharmacy-production-unit.aspx).

Investigational Medicinal Product management

l Bovine lactoferrin was packaged into 25-ml opaque pharmacy pots (fill equivalent to 375 mg per pot)

at the trials pharmacy in Newcastle Royal Infirmary, Newcastle upon Tyne, UK. Boxes containing 24

identically numbered pots were labelled with the same pack identification (ID) number to indicate that

they all belonged to the same treatment course. At randomisation, infants were allocated a study

number and a pack ID number; the study number was added to the label of the allocated pack with

the infant’s name and date of birth for checking before each administration of the IMP. Lactoferrin

powder was stable within unopened pots and could be stored at room temperature. When the infant

completed the course of treatment, any unused IMP pots were retained for accounting and destruction

by the site pharmacist.l Sucrose was processed, packaged and distributed as for bovine lactoferrin.

Investigational Medicinal Product prescription and preparationLactoferrin and sucrose were prescribed at a dose of 150 mg/kg body weight per day (up to a maximum of

300 mg/day). The IMP was prepared by neonatal nurses or clinicians on the neonatal unit in the unit milk

kitchen or other appropriate area determined locally. The IMP powder was prepared for administration by

mixing in sterile water plus expressed breast or formula (see Appendix 3).

The IMP was administered once daily by nasogastric or orogastric tube or orally once the enteral feed

volume was > 12 ml/kg/day and continued until 34 weeks’ postmenstrual age. Some small infants may

have had the dose split at the discretion of the responsible clinical team. A maximum of 70 days of

treatment was given.

All other aspects of care, including the timing of the commencement of enteral feeds and the type of milk

feed used, were as per local policy, practice and discretion.



7

https://www.tatua.com/specialty-nutritionals-ingredients/lactoferrin/

https://www.tatua.com/specialty-nutritionals-ingredients/lactoferrin/

https://www.britishsugar.co.uk/

https://www.newcastle-hospitals.org.uk/services/Pharmacy_services_newcastle-specials-pharmacy-production-unit.aspx

https://www.newcastle-hospitals.org.uk/services/Pharmacy_services_newcastle-specials-pharmacy-production-unit.aspx

Randomisation

Randomisation of participants to receive either lactoferrin or sucrose was managed via a secure web-based

randomisation facility hosted by the NPEU CTU, University of Oxford, Oxford, UK. Telephone assistance

and randomisation back-up was available at all times.

To confirm eligibility, investigators needed to supply gestational age, sex and time of birth. To proceed to

randomisation, investigators needed to confirm that signed informed consent was available. Infants were

allocated to the lactoferrin versus sucrose groups in the ratio of 1 : 1 using a minimisation algorithm to

ensure balance between the groups with respect to the recruiting site (neonatal unit), sex, single versus

multiple births and gestational age in completed weeks. Twins or higher order multiple births were

randomised individually.

Allocation concealment and blindingParticipating infants were randomly allocated a numbered pack containing either the lactoferrin or the

placebo and allocated a unique study number. Parents, clinicians, investigators and outcomes assessors

were unaware of the allocated treatment groups.

Stopping Investigational Medicinal ProductAdministration of the trial IMP may have been stopped temporarily. Missed doses did not necessitate the

removal of an infant from the trial. Data continued to be collected as per protocol if the trial medication

was stopped temporarily or permanently in order to facilitate an unbiased treatment comparison via an

intention-to-treat analysis.

MaskingBovine lactoferrin has a pale pink/brown tinge, whereas sucrose was very light brown. The opaque containers

did not allow the dry IMP to be seen unless the sealed stopper was removed intentionally. The lactoferrin

powder had similar granularity to sucrose so that when the dry IMP was shaken within the opaque, sealed

pots it was not possible to distinguish lactoferrin from sucrose by the sound generated. Mixing the IMP

with sterile water plus breast milk or formula generated foam that settled within 30 minutes after shaking.

When the mixed IMP was removed in a syringe with a purple plunger, the pink tinge to the lactoferrin was

disguised by the colour of the breast milk or formula, which often resulted in a light brown colour (and this

varied markedly between batches of milk). As lactoferrin was more likely than sucrose to retain a light pink

tinge, all sites were supplied with a laminated picture of a range of possible colours for the IMP mixture in

syringes, and it was stressed that this applied to both lactoferrin and sucrose.

Clinicians were able to request knowledge of the treatment allocation to guide the clinical management of

the participant if it was deemed to be an emergency situation. In such instances, a single-use access code

was provided in a sealed envelope and the participant’s allocation was unmasked via the randomisation

website.

Internal pilot

An internal pilot study was conducted in six neonatal centres within the Northern Region and Yorkshire

Neonatal Networks (‘operational delivery networks’) to test whether or not the components and processes

of the study worked together and ran smoothly. The main aims of the pilot were to:

1. confirm that regulatory processes were in order

2. ensure that the randomisation process was acceptable and effective

3. demonstrate efficient intervention and placebo preparation and distribution

4. determine that the anticipated acceptance rate (40% of eligible infants) was achievable

METHODS


8

5. determine whether or not the projected recruitment rate was realistic – we set a target of a total of

4, 6 and 8 infants recruited in months 1, 2 and 3, respectively, expecting to reach a ‘steady state’ of a

total of 10 infants (two per month per centre) by month 4 of the pilot phase

6. evaluate the delivery, management and acceptability (to families and staff) and ease of preparation

and administration

7. assess the processes for collecting clinical outcomes and event rates and to determine that the

predicted retention rate (> 95% of recruited infants) was attainable.

The decision to progress with the main trial was made in consultation with the Trial Steering Committee

(TSC) and the funder.

The internal pilot phase was followed by a 3-year main recruitment phase in 37 recruiting centres

(see Appendix 1).

Outcomes

Primary outcomeThe number of infants who experience at least one episode of microbiologically confirmed (Box 3) or

clinically suspected (Box 4) late-onset infection from trial entry until hospital discharge.

Secondary outcomes

l Microbiologically confirmed infection (see Box 3).l All-cause mortality prior to hospital discharge.l Necrotising enterocolitis: Bell’s stage II or III (see Appendix 4).52

l Severe ROP treated medically or surgically.53

l Bronchopulmonary dysplasia: when the infant is still receiving mechanical ventilator support or

supplemental oxygen at 36 weeks’ postmenstrual age.54

l A composite of invasive infection, major morbidity (NEC, ROP or BPD) and mortality.l Total number of days of administration of antimicrobials per infant from trial entry until 34 weeks’

postmenstrual age.l Total length of stay until discharge home.l Length of stay in (1) intensive care, (2) high-dependency and (3) special-care settings (see Appendix 5).

BOX 3 Definition of microbiologically confirmed late-onset infection

Microbiological culture from blood or CSF sampled aseptically > 72 hours after birth of any of the following:

l potentially pathogenic bacteria (including coagulase-negative Staphylococcus species but excluding

probable skin contaminants, such as diphtheroids, micrococci, propionibacteria or a mixed flora)

l fungi.

AND treatment, or clinician intention to treat, for ≥ 5 days with intravenous antibiotics (excluding antimicrobial

prophylaxis) after investigation was undertaken. If the infant died or was discharged or transferred prior to the

completion of 5 days of antibiotics this condition would still be met if the intention was to treat for ≥ 5 days.

Adapted from the UK Neonatal Infection Surveillance Network case definition.2,3



9

Sample size

The sample size estimate was informed by a range of plausible primary outcome control event rates (CERs)

from 18% to 24%, based on surveillance reports from Europe, North America and Australasia (Table 1).2,7,8,10,12

In summary, with 90% power and a two-sided 5% significance level, to detect an absolute risk reduction

(ARR) of 5–5.8% (relative risk reduction of between 24% and 28%) would require a total of up to 2200

participants if the CER was 18%, 2070 if the CER was 21% and 2076 if the CER was 24%. This target sample

size of 2200 allowed for an anticipated loss to follow-up of up to 5%. This sample size was sufficient to

exclude important effects on secondary outcomes with 90% power [e.g. a 7% ARR in antibiotic exposure

(from 45% to 38%)].

The participating recruiting neonatal units were estimated to admit 60 very preterm infants per annum on

average. Based on 40% recruitment, 30 units were estimated to be able to recruit a total sample size of

up to 2160 infants over 3 years (an average of two infants per unit per month).

BOX 4 Definition of clinically suspected late-onset infection

Absence of positive microbiological culture, or culture of a mixed microbial flora or of likely skin contaminants

(diphtheroids, micrococci, propionibacteria) only

AND treatment, or clinician intention to treat, for ≥ 5 days with intravenous antibiotics (excluding antimicrobial

prophylaxis) after the above investigation was undertaken for an infant who demonstrates three or more of the

following clinical or laboratory features of invasive infection:

l increase in oxygen requirement or ventilatory support

l increase in frequency of episodes of bradycardia or apnoea

l temperature instability

l ileus or enteral feeds intolerance or abdominal distension

l reduced urine output to < 1ml/kg/hour

l impaired peripheral perfusion (capillary refill time of > 3 seconds, skin mottling or core–peripheral

temperature gap > 2 °C)

l hypotension (clinician defined as needing volume or inotrope support)

l ‘irritability or lethargy or hypotonia’ (clinician defined)

l increase in serum C-reactive protein levels to > 15mg/l or procalcitonin level of ≥ 2 ng/ml

l white blood cells count of < 4 × 109/l or > 20 × 109/l

l cells/l or platelet count of < 100 × 109/l

l glucose intolerance (blood glucose levels of < 40mg/dl or > 180 mg/dl)

l metabolic acidosis (base excess of < –10mmol/l or lactate level of > 2mmol/l).

Adapted from the European Medicines Agency consensus criteria and predictive model.50,51

TABLE 1 Participants required per arm by CER

Control eventrate (%)

Treatment groupevent rate (%)

Absolute riskreduction (%)

Relative riskreduction (%)

Numberrequiredper arm

Total samplesize required

24 18.2 5.8 24 1038 2076

21 15.5 5.5 26 1035 2070

18 13.0 5.0 28 1099 2200

METHODS


10

Statistical analyses

Demographic factors and clinical characteristics at randomisation were summarised with counts (percentages)

for categorical variables, mean [standard deviation (SD)] for normally distributed continuous variables or

median [interquartile range (IQR)] for other continuous variables.

Outcomes for participants were analysed in the groups to which they were assigned regardless of deviation

from the protocol or treatment received. Comparative analyses calculated the relative risk ratio (RR) with 95%

confidence interval (CI) for the primary outcome (99% CIs for all other dichotomous outcomes), the mean

difference (99% CI) for normally distributed continuous outcomes or the median difference (99% CI) for

skewed continuous variables.

The groups were compared using regression analysis adjusting for the minimisation factors (recruiting

centre, sex, weeks’ gestation at birth and single vs. multiple births) to account for the correlation between

treatment groups introduced by balancing the randomisation. We used random-effects models with centre

fitted as a random effect and mother’s ID number nested within this to take account of clustering within

centre and within multiples. The other minimisation factors were fitted as fixed effects, with sex and

multiplicity of birth included as binary variables and gestational age modelled as a continuous variable.

The crude unadjusted and adjusted estimates were calculated with the primary inference to be based on

the adjusted analysis.

The consistency of the effect of lactoferrin supplementation on the primary outcome across specific

subgroups of infants was assessed using the statistical test of interaction. Prespecified subgroups were

(1) completed weeks of gestation at birth and (2) infants given maternal or donated expressed breast milk

versus formula versus both human milk and formula during the trial period (received on > 50% of days

on which infant is fed enterally until developing late-onset infection or NEC, dying or reaching 34 weeks’

postmenstrual age).

Data collection

All of the outcome data for this trial were routinely recorded clinical items that could be obtained from the

clinical notes or local microbiology laboratory records. Information was collected using the data collection

forms (see Appendix 6).

A ‘blinded end-point review committee’, masked to participant allocation, reviewed all case report forms

(CRFs) reporting episodes of late-onset infection or necrotising enterocolitis or other gastrointestinal pathology.

Two members independently assessed adherence to case definitions and resolved any disagreements or

discrepancies by discussion or referral to a third committee member or both. Persisting uncertainties were

discussed with the site PI or research nurse or both until resolved.

Adverse event reporting

Some adverse events were foreseeable (expected) because of the nature of the participant population,

and their routine care and treatment. No adverse drug reactions were expected from bovine lactoferrin.

Consequently, only those adverse events (or reactions) identified as serious were recorded for this trial

(see Appendix 7).

Expected serious adverse events (SAEs) were recorded on the CRFs. All other SAEs were reported by trial

sites to the NPEU CTU within 24 hours of the event being recognised. Information was recorded on a

SAE reporting form and faxed to the NPEU CTU. Additional information (follow-up of or corrections to the

original case) needed to be detailed on a new SAE form and faxed to the NPEU CTU. A standard operating

procedure (SOP) outlining the reporting procedure for clinicians was provided with the SAE form and in



11

the trial handbook. The NPEU CTU processed and reported the event as specified in its own SOPs. All SAEs

were reviewed by the Data Monitoring Committee (DMC) at regular intervals throughout the trial. The CI

informed all investigators of information that could affect the safety of participants.

Suspected unexpected serious adverse reactionsSuspected unexpected serious adverse reactions (SUSARs) were reported to the MHRA and the approving

Research Ethics Committee (REC) within 7 days, if life-threatening, and within 15 days for other SUSARs.

In addition, a copy of the SUSAR form was forwarded to the chairperson of the DMC. The chairperson

was provided with details of all previous SUSARs with their unmasked allocation. The chief investigator

informed all investigators of any issues raised in a SUSAR that could affect the safety of participants.

Development safety update reportIn addition to the expedited reporting above, the chief investigator submitted, once a year throughout the

clinical trial, or on request, a development safety update report to the Competent Authority, the Ethics

Committee and the sponsor.

Economic analysis (planned)

We planned to combine the health service resources used during an infant’s hospital admission with

clinical effectiveness data to conduct an economic evaluation to assess whether or not the intervention

was likely to be cost-effective over the time horizon of the trial period. If appropriate, we intended to

synthesise the costs and consequences of the intervention to generate an incremental cost-effectiveness

ratio to inform any adoption decision. We planned to use regression models to allow for differences in

prognostic variables, principally gestational age bands, and other sources of heterogeneity and to assess

differences in the probable cost-effectiveness between the groups.

The primary outcome for an economic analysis was the incidence of late-onset invasive infection. As invasive

infection is linked closely to morbidity and mortality, it is likely that the consequences will continue to appear

over a longer time frame and may have an impact on both duration and quality of life. Therefore, as a second

analysis, we intended to develop an economic model to account for projected longer-term costs and effects

and to estimate the additional cost per quality-adjusted life-year gained of lactoferrin compared with placebo.

Governance and monitoring

At least one site initiation visit was conducted at all recruiting sites. The trial research nurse and the chief

investigator or a delegated co-investigator provided structured training for site investigators, local research

nurses and other clinical staff, including the site pharmacy team responsible for IMP management. Training

focused on approaches to consent, protocol processes and governance requirements. These visits were

supported with bespoke written and online training material available to all staff via the trial website

(www.npeu.ox.ac.uk/elfin/training). Staff in continuing care sites did not have initiation visits unless requested

but were directed to online training and access support from the chief investigator and trial research nurse

as needed.

Ongoing monitoring included review of investigator site files, delegation logs, staff qualifications and

training (good clinical practice certificates, curricula vitae) and pharmacy documentation. Quality assurance

was achieved by following data management procedures at the study data centre and data monitoring at

trial sites. Further site monitoring or audit was conducted if central monitoring exercises raised concern

about patterns of recruitment or data reporting. This monitoring approach was justified by the level of risk

associated with the trial and the intervention.

METHODS


12

https://www.npeu.ox.ac.uk/elfin/training

Data management was undertaken in accordance with NPEU CTU SOPs and a prespecified management

plan. Data monitoring included the review of consent forms and participant eligibility. Additional data

validation checks were carried out periodically, with data queries issued to study sites for resolution. Prior

to database lock, final data validation checks were carried out and questions were resolved by discussion

with the site PI or local research nurse, when possible.

During the trial, the study statisticians produced reports for the TSC and independent DMC. Issues of data

quality identified by study statisticians were reported to study data management staff and queried when

appropriate or included in future routing data validation checks, or both. TSC and DMC meetings provided

opportunities for the external, independent review of summary data, with additional feedback on potential

data quality issues being incorporated into ongoing data quality checks.

Summary of changes to the study protocol

A summary of the changes made to the original protocol is presented in Appendix 8.



13

Chapter 3 Results

Recruitment and retention

Recruitment and retention to the trial are summarised in the flow chart (Figure 1).

The internal pilot was undertaken in six neonatal units between May 2014 and April 2015. In total,

90 infants were recruited to participate. The main trial recruited infants from 37 neonatal units (including

the six pilot sites) from July 2015 to September 2017 (when the recruitment target was reached). The trial

randomised 2203 infants in total:

l 1099 infants were allocated to receive bovine lactoferrinl 1104 infants were allocated to receive the sucrose placebo.

Four infants had consent withdrawn or unconfirmed. In total, 1098 infants in the lactoferrin group and

1101 in the placebo group were included in the intention-to-treat analyses (see Appendix 9).

Demographic and other baseline characteristics

The baseline characteristics and other demographic features of participating infants were well balanced

between the two treatment allocation groups (Table 2). The median gestation age was 29 weeks in both

groups (37% aged < 28 weeks). The median birthweight was 1126 g in the lactoferrin group and 1143 g

in the placebo group. Overall, 91% of infants were exposed to antenatal corticosteroid, 57% were born

via caesarean section, 25% were born following rupture of maternal amniotic membranes for > 24 hours,

Infants randomised(n = 2203)

(1863 women)

Infants allocated to lactoferrin(n = 1099)

• Received no doses, n = 21 (1.9%)• Randomised in error, n = 8 (0.7%) • Randomised at > 72 hours, n = 7 • Consent not confirmed, n = 1• Withdrawn, n = 20 (1.8%)• Consent to use data remains,a n = 20

Babies included in analysis ofoutcomes at discharge

(n = 1098)

• Consent not confirmed, n = 1• Outcome status not known,b n = 21

Babies included in analysis ofoutcomes at discharge

(n = 1101)

• Withdrawn consent to use data, n = 3• Outcome status not known,b n = 24

All

oca

tio

nA

naly

sis

at

dis

charg

e

Infants allocated to placebo(n = 1104)

• Received no doses, n = 31 (2.8%)• Received ≥ 1 dose of lactoferrin, n = 1• Randomised in error, n = 1 (0.1%) • Randomised at > 72 hours, n = 1• Withdrawn, n = 15 (1.4%)• Withdrawn consent to use data, n = 3• Consent to use data remains,a n = 12

FIGURE 1 Flow of participants through the trial. a, Included in the analysis when data are available; b, included inanalysis except when knowledge of discharge or discharge date is required.



15

TABLE 2 Infant and maternal characteristics at randomisation

Characteristic

Trial group

Lactoferrin (n= 1098) Placebo (n= 1101)

Number of centres, n 37 37

Male sex, n/N (%) 575/1098 (52.4) 578/1099 (52.6)

Missing, n 0 2

Infant age at randomisation in days

Median (IQR) 2 (2–3) 2 (2–3)

Birthweight (g)

Mean (SD) 1125.9 (356.2) 1143.3 (367.1)

< 500, n (%) 8/1098 (0.7) 7/1101 (0.6)

500 to 749, n (%) 172/1098 (15.7) 172/1101 (15.6)

750 to 999, n (%) 254/1098 (23.1) 244/1101 (22.2)

1000 to 1249, n (%) 268/1098 (24.4) 255/1101 (23.2)

1250 to 1499, n (%) 199/1098 (18.1) 199/1101 (18.1)

≥ 1500, n (%) 197/1098 (17.9) 224/1101 (20.3)

Birthweight < 10th centile for gestational age, n/N (%) 175/1097 (16.0) 177/1098 (16.1)

Missing, n 1 3

Gestation at delivery (completed weeks)

Median (IQR) 29 (27–30) 29 (27–30)

< 23, n/N (%) 1/1098 (0.1) 1/1101 (0.1)

23+0 to 23+6, n/N (%) 33/1098 (3.0) 31/1101 (2.8)

24+0 to 24+6, n/N (%) 73/1098 (6.6) 76/1101 (6.9)

25+0 to 25+6, n/N (%) 73/1098 (6.6) 73/1101 (6.6)

26+0 to 27+6, n/N (%) 227/1098 (20.7) 221/1101 (20.1)

28+0 to 29+6, n/N (%) 315/1098 (28.7) 319/1101 (29.0)

30+0 to 31+6, n/N (%) 376/1098 (34.2) 380/1101 (34.5)

Mother’s age at randomisation (years)

Mean (SD) 30.3 (6.1) 30.4 (6.0)

Multiple birth, n/N (%) 350/1098 (31.9) 346/1101 (31.4)

Caesarean section delivery, n/N (%) 635/1098 (57.8) 616/1101 (55.9)

Membranes ruptured before labour, n/N (%) 422/1093 (38.6) 428/1097 (39.0)

Missing, n 5 4

Membranes ruptured > 24 hours before delivery, n/N (%) 286/1092 (26.2) 264/1096 (24.1)

Missing, n 6 5

Mother received antenatal corticosteroids, n/N (%) 998/1093 (91.3) 997/1099 (90.7)

Missing, n 5 2

Infant heart rate of > 100 b.p.m. at 5 minutes, n/N (%) 995/1090 (91.3) 1010/1093 (92.4)

Missing, n 8 8

RESULTS


16

and 12% had evidence of absent or reverse end diastolic flow in the fetal umbilical artery. The allocation

arms were well-balanced in individual recruiting sites as per the minimisation algorithm (see Appendix 10).

Adherence

A total of 35 (1.6%) infants discontinued the intervention early: 20 in the lactoferrin group and 15 in the

sucrose group. This includes a small number of infants who in the early stages of the trial discontinued

the intervention because they were transferred to a hospital that did not have the regulatory approvals to

administer the intervention. Parental consent remained for data collection for intention-to-treat analyses

for 32 out of the 35 infants.

Adherence was high for infants who continued to receive the IMP (Table 3). The median percentage of

days when an IMP dose was not given or not recorded was 4% in both treatment groups, and 0% of

days in both groups for the dose not given or not recorded when those days where feeds were stopped

or withheld for > 4 hours (for clinical reasons) were excluded. The median difference between expected

dose and actual dose per day was 7 mg/kg/day lower in both groups and was 1 mg/kg/day (lactoferrin)

or 2 mg/kg/day (sucrose) lower excluding those days where enteral feeds were stopped or withheld for

> 4 hours.

Outcomes

The estimates of effect for the primary and secondary outcomes are presented in Table 4.

Primary outcomeData were available for 2182 infants (99%). In the lactoferrin group, 316 out of 1093 (28.9%) infants

acquired a late-onset infection versus 334 out of 1089 (30.7%) infants in the control (placebo) group

(adjusted RR 0.95, 95% CI 0.81 to 1.10).

TABLE 2 Infant and maternal characteristics at randomisation (continued )

Characteristic

Trial group


Infant temperature on admission (°C)

Mean (SD) 36.9 (0.7) 37 (0.7)

Missing, n 4 10

Infant worst base excess within first 24 hours of birth

Mean (SD) –6.2 (3.9) –6.3 (3.8)

Missing, n 9 12

Infant ventilated via endotracheal tube at randomisation, n/N (%) 338/1098 (30.8) 357/1101 (32.4)

Infant had absent or reverse end diastolic flow, n/N (%) 134/1079 (12.4) 130/1081 (12.0)

Missing, n 19 20

b.p.m., beats per minute.Bold indicates minimisation factors.Unless otherwise stated, the table gives the percentages of babies with data in that arm of the trial who had (or whosemother had) the stated characteristic.



17

TABLE 3 Adherences measures

Measure

Trial group

Lactoferrin (n= 1007)a Placebo (n= 1011)a

Percentage of days dose not given or not recordedb

Median (IQR) 4 (0 to 18.18) 4 (0 to 16.22)

Range 0 to 100 0 to 100

Missing, n 10 13

Percentage of days dose not given or not recorded, excluding dayswhere feeds were stopped or withheld for > 4 hoursb

Median (IQR) 0 (0 to 5.71) 0 (0 to 5.56)

Range 0 to 100 0 to 100

Missing, n 11 13

Difference between expected dose and actual dose per day (mg/kg/day)c

Median (IQR) –7 (–29 to 0) –7 (–27 to 0)

Range –150 to 253 –150 to 88

Missing, n 10 13

Difference between expected dose and actual dose per day, excludingdays where feeds were stopped or withheld for > 4 hours (mg/kg/day)c

Median (IQR) –1 (–11 to 0) –2 (–11 to 0)

Range –150 to 271 –150 to 88

Missing, n 11 13

a Includes only infants who have completed at least one feed log, and doses are exclusively recorded on version 2 of thedaily dosing log.

b The ratio of the number of days the dose was not given or recorded over the number of days in the expectedtreatment window.

c The average difference between the expected dose and actual dose over every day of treatment window. Whennecessary, the working weight is imputed using last observation carried forward.

TABLE 4 Primary and secondary outcomes

Outcome

Trial group

UnadjustedRR (CI)a,b

AdjustedRR (CI)a,b,c p-valued

Lactoferrin(n= 1098)

Placebo(n= 1101)

Microbiologically confirmed or clinicallysuspected late-onset infection, n/N (%)

316/1093(28.9)

334/1089(30.7)

0.94(0.83 to 1.07)

0.95(0.86 to 1.04)

0.233

Missing, n 5 12

Microbiologically confirmed late-onsetinfection, n/N (%)

190/1093(17.4)

180/1089(16.5)

1.05(0.82 to 1.34)

1.05(0.87 to 1.26)

0.490

Missing, n 5 12

All-cause mortality, n/N (%) 71/1076(6.6)

68/1076(6.3)

1.04(0.69 to 1.59)

1.05(0.66 to 1.68)

0.782

Missing, n 22 25

NEC (Bell’s stage II or III), n/N (%) 63/1085(5.8)

56/1084(5.2)

1.12(0.71 to 1.77)

1.13(0.68 to 1.89)

0.538

Missing, n 13 17

RESULTS


18

TABLE 4 Primary and secondary outcomes (continued )

Outcome

Trial group

UnadjustedRR (CI)a,b

AdjustedRR (CI)a,b,c p-valued


Placebo(n= 1101)

Severe ROP treated medically orsurgically, n/N (%)

64/1080(5.9)

72/1080(6.7)

0.89(0.58 to 1.35)

0.89(0.62 to 1.28)

0.420

Missing, n 18 21

BPD at 36 weeks’ postmenstrual age,n/N (%)

358/1023(35.0)

355/1027(34.6)

1.01(0.87 to 1.18)

1.01(0.90 to 1.13)

0.867

Died before 36 weeks’postmenstrual age

64 60

Missing, n 11 14

Infection, NEC, ROP, BPD or mortality,n/N (%)

525/1092(48.1)

521/1094(47.6)

1.01(0.90 to 1.13)

1.01(0.94 to 1.08)

0.743

Missing, n 6 7

Total number of days of administrationof antimicrobials from commencementof IMP until 34 weeks’ postmenstrualage

Median (IQR) 2 (0–8) 3 (0–8) 0 (0 to 0) 0 (–1 to 1) 0.625

Missing, n 39 44

Length of hospital stay (days) todischarge

Median (IQR) 59 (40–85) 58 (40–84) 1 (–2 to 4) 1 (–1 to 3) 0.446

Missing, n 95 97

Days in level 1 (intensive) care

Median (IQR) 8 (4–16) 8 (4–16) 0 (–1 to 1) 0 (–1 to 1) 0.963

Missing, n 87 66

Days in level 2 (high-dependency) care

Median (IQR) 10 (3–30) 9 (3–29) 0 (–1 to 1) 1 (–1 to 3) 0.420

Missing, n 83 60

Days in level 3 (special) care

Median (IQR) 29 (21–39) 30 (22–39) –1 (–2 to 1) –1 (–3 to 1) 0.216

Missing, n 75 55

a Risk ratios for binary outcomes and median differences for continuous outcomes.b 95% CI for microbiologically confirmed or clinically suspected late-onset invasive infection, 99% CI for all other outcomes.c Adjusted for minimisation factors: collaborating hospital, sex, gestational age at birth vs. single or multiple births,

when technically possible.d p-value for testing whether or not adjusted RR is equal to 1 or adjusted median difference is equal to 0.



19

Secondary outcomesThere were no significant differences in secondary outcomes: microbiologically confirmed infection

(RR 1.05, 99% CI 0.80 to 1.37), mortality (RR 1.05, 99% CI 0.68 to 1.63), NEC (RR 1.13, 99% CI 0.70

to 1.82), ROP (RR 0.89, 99% CI 0.57 to 1.40), BPD (RR 1.01, 99% CI 0.83 to 1.22), or a composite of

infection, major morbidity and mortality (RR 1.01, 99% CI 0.86 to 1.18). There were no differences in the

number of days of administration of antimicrobials until 34 weeks’ postmenstrual age, or in length of stay

in hospital, or length of stay in intensive care, high-dependency care or special-care settings.

Subgroup analysesSubgroup analyses did not show any significant interactions for completed weeks’ gestation at birth or

type of enteral milk received (Table 5 and Figure 2).

Economic analysis

Given the absence of any effects on infant- or family-important outcomes (clinical effectiveness), and with

the approval of the DMC and TSC, we did not undertake the proposed within-trial economic analyses or

modelling (protocol amendment submitted).

Safety and adverse events

Table 6 summarises reported adverse events (definitions of adverse reactions and events are presented in

Appendix 7).

There were 16 SAEs reported for infants in the lactoferrin group (six severe) and 10 for infants in the sucrose

group (three severe). No infant had more than one reported event. Two SAEs, both in the lactoferrin group,

were assessed as being ‘possibly related’ to the trial intervention: one case of blood in stool (expected) and

one death following intestinal perforation likely associated with NEC (SUSAR). The remaining 24 SAEs were

considered to be unrelated to the trial intervention.

TABLE 5 Subgroup analyses for confirmed or suspected late-onset infection

Late-onsetinfection

Trial group

Unadjusted RR(95% CI)

Adjusted RRa

(95% CI) p-valueb


Placebo(n= 1101)

Gestational age at delivery (completed weeks), n/N (%) 0.273

< 24 25/34 (73.5) 27/31 (87.1) 0.84 (0.66 to 1.07) 0.91 (0.69 to 1.20)

24 46/73 (63.0) 56/75 (74.7) 0.84 (0.68 to 1.05) 0.84 (0.69 to 1.03)

25 45/73 (61.6) 44/73 (60.3) 1.02 (0.78 to 1.34) 1.03 (0.73 to 1.45)

26 to 27 107/227 (47.1) 99/220 (45.0) 1.05 (0.86 to 1.28) 1.04 (0.85 to 1.28)

28 to 29 69/311 (22.2) 72/316 (22.8) 0.97 (0.73 to 1.30) 0.98 (0.74 to 1.29)

≥ 30 24/375 (6.4) 36/374 (9.6) 0.66 (0.40 to 1.10) 0.66 (0.42 to 1.03)

Type of milk, n/N (%) 0.400

Breast only 99/315 (31.4) 83/291 (28.5) 1.10 (0.87 to 1.40) 1.03 (0.88 to 1.21)

Mixed 199/707 (28.1) 228/710 (32.1) 0.88 (0.75 to 1.03) 0.89 (0.79 to 1.01)

Formula only 10/53 (18.9) 12/60 (20.0) 0.94 (0.45 to 2.00) 1.06 (0.58 to 1.91)

Missing, n 18 29

a Adjusted for minimisation factors: recruiting site, sex, gestational age at birth (completed weeks) and single vs. multiple birth.b p-value for test for interaction from adjusted model.

RESULTS


20

0.25 0.5 1 2 4

Favours lactoferrin Favours placebo

SubgroupLactoferrin(n/N)

Placebo(n/N)

Risk ratio(95% CI) p-value

Gestational age

< 24 weeks

24+0 to 24+6 weeks

25+0 to 25+6 weeks

26+0 to 27+6 weeks

28+0 to 29+6 weeks

≥ 30 weeks

25/34

46/73

45/73

107/227

69/311

24/375

27/31

56/75

44/73

99/220

72/316

36/374

0.91 (0.69 to 1.20)

0.84 (0.69 to 1.03)

1.03 (0.73 to 1.45)

1.04 (0.85 to 1.28)

0.98 (0.74 to 1.29)

0.66 (0.42 to 1.03)

0.273

99/315

199/707

10/53

83/291

228/710

12/60

1.03 (0.88 to 1.21)

0.89 (0.79 to 1.01)

1.06 (0.58 to 1.91)

0.400Type of milk

Breast only

Mixed

Formula only

FIGURE 2 Subgroup analyses for confirmed or suspected late-onset invasive infection.

TABLE 6 List of SAEs reported by randomisation group

Trial group SAEAge(days) Brief description of event Severity

Relatedto trial

Lactoferrin(n = 1098)

1 12 Meconium ileus following one dose of IMP. Resolved withlaparotomy, no bowel removed

Moderate No

2 30 Two episodes of clinical seizures, resolved with brief course ofanticonvulsant

Moderate No

3 59 Cluster of seizures, probably related to severe Gram-negativebacteraemia and sepsis (ultimately fatal)

Severe No

4 12 Episode of supraventricular tachycardia, resolved withadenosine and propranolol

Mild No

5 49 Metabolic acidosis (likely renal tubular acidosis), resolved withsodium bicarbonate

Severe No

6 20 Episode of supraventricular tachycardia, resolved with facecooling

Mild No

7 19 Suspected NEC Moderate No

8 18 Cluster of clinical seizures, resolved with magnesium sulphateand course of phenobarbital

Mild No

9 81 Infective exacerbation of chronic lung disease, resolved withantibiotics and corticosteroids

Severe No

10 17 Large inferior vena caval thrombus Moderate No

11 68 Acute airway obstruction, resolved with respiratory support Severe No

12 44 Aspiration pneumonitis resolved with respiratory support Severe No

13 21 Blood in stool, unknown cause, resolved spontaneously Moderate Possibly(expected)

14 19 Haemolytic anaemia, unknown cause, resolved spontaneously Mild No

continued



21

Post hoc analyses

1. Post hoc exploratory analyses did not show any differential effects depending on the infecting

micro-organism identified for the outcome ‘microbiologically confirmed late-onset infection’ (Table 7

and Box 5).

2. Post hoc exploratory analyses did not show any between-group differences in the risk of having more

than one episodes of infection (Table 8).

3. Post hoc exploratory analyses did not show any differential effects for the primary outcome depending

on whether infants had or had not received probiotics as part of their routine care (Table 9).

TABLE 6 List of SAEs reported by randomisation group (continued )

Trial group SAEAge(days) Brief description of event Severity

Relatedto trial

15 10 Death following intestinal perforation secondary to NEC Severe Possibly(SUSAR)

16 27 Death attributed to Gram-negative bacteraemia Severe No

Placebo(n = 1101)

1 61 Rib fracture secondary to osteopenia of prematurity, resolvedwith supportive care and nutrient supplementation

Moderate No

2 50 Superior sagittal sinus non-occlusive thrombus, resolved withheparin (6 weeks of treatment)

Moderate No

3 48 Hyperammonaemia, unknown cause, resolved with course ofsodium benzoate

Moderate No

4 36 Death attributed to infection and sepsis Severe No

5 24 Episode of tachycardia and ectopic beats, resolved with facecooling and reduction in caffeine dose

Mild No

6 37 Death secondary to exacerbation of chronic lung disease(severe BPD)

Severe No

7 26 Death attributed to severe BPD Severe No

8 57 S. aureus bacteraemia and osteomyelitis, resolved withantibiotics

Moderate No

9 22 Episode of supraventricular tachycardia, resolved withadenosine

Moderate No

10 6 Episode of supraventricular tachycardia, resolved with carotidmassage and adenosine

Mild No

RESULTS


22

BOX 5 Classification of micro-organisms

1. Staphylococcus epidermidis.

2. Staphylococcus capitis.

3. Other coagulase-negative Staphylococci.

4. S. aureus.

5. Enterococcus faecalis.

6. Group B streptococci.

7. Enterococcus sp. (other).

8. Streptococcus sp. (other).

9. Micrococcus sp.

10. Bacillus sp.

11. Diphtheroids.

12. Streptococcus pneumoniae.

13. Propionibacterium acnes.

14. Listeria monocytogenes.

15. Other Gram-positive bacteria.

16. Escherichia coli.

17. Klebsiella sp.

18. Enterobacter sp.

19. Pseudomonas sp.

20. Serratia sp.

21. Coliforms (other).

22. Acinetobacter sp.

23. Citrobacter sp.

24. Burkholderia sp.

25. Haemophilus sp.

26. Other Gram-negative bacteria.

27. Candida albicans.

28. Candida sp. (other).

29. Other fungi.

30. Other organisms.

1–15, Gram positive; 1–3, coagulase-negative Staphylococcus group; 16–26, Gram negative; 27–29, fungi;

30, other.

TABLE 7 Microbiologically confirmed late-onset infection by classification of micro-organism

Classification of micro-organism

Trial group


Microbiologically confirmed late-onset invasive infectionfrom trial entry until hospital discharge, n/N (%)

190/1093 (17.4) 180/1089 (16.5)

At least one Gram-positive organism confirmed, n/N (%) 153/1093 (14.0) 147/1089 (13.5)

At least one CoNS group organism, n/N (%) 122/1093 (11.2) 111/1089 (10.2)

At least one Gram-negative organism confirmed, n/N (%) 46/1093 (4.2) 39/1089 (3.6)

At least one fungal organism confirmed, n/N (%) 3/1093 (0.3) 2/1089 (0.2)

At least one other organism confirmed, n/N (%) 3/1093 (0.3) 2/1089 (0.2)

Missing, n 5 12

CoNS, coagulase-negative Staphylococci.



23

TABLE 8 Number of episodes of confirmed or suspected sepsis

Number of episodes

Trial group


Microbiologically confirmed or clinically suspected sepsis, n/N (%)

None 777/1093 (71.1) 755/1089 (69.3)

1 258/1093 (23.6) 279/1089 (25.6)

2 46/1093 (4.2) 39/1089 (3.6)

3 9/1093 (0.8) 13/1089 (1.2)

4 3/1093 (0.3) 2/1089 (0.2)

5 0/1093 (0.0) 1/1089 (0.1)

Missing, n 5 12

Microbiologically confirmed infection, n/N (%)

None 903/1093 (82.6) 909/1089 (83.5)

1 162/1093 (14.8) 155/1089 (14.2)

2 24/1093 (2.2) 23/1089 (2.1)

3 3/1093 (0.3) 2/1089 (0.2)

4 1/1093 (0.1) 0/1089 (0.0)

TABLE 9 Late-onset infection from trial entry until hospital discharge by exposure to probiotics

Trial group


Any record of probiotics being given, n/N (%)

Yes 99/354 (28.0) 97/329 (29.5)

No 208/728 (28.6) 227/749 (30.3)

Missing,a,b n 16 23

a In the lactoferrin trial group, 15 babies had unknown probiotic use; nine had an episode of late-onset infection, two didnot and four were unknown. One other baby with a record of probiotic use had unknown infection status.

b In the placebo trial group, 19 babies had unknown probiotic use; 10 had an episode of late-onset infection, one did notand eight were unknown. Four other babies with a record of probiotic use had unknown infection status.

RESULTS


24

Chapter 4 Discussion and conclusions

Summary of main findings

The ELFIN trial shows that enteral lactoferrin supplementation (150 mg/kg/day until 34 weeks’ postmenstrual

age) does not reduce the risk of late-onset infection, other morbidity or mortality in very preterm infants.

This finding contradicts the existing evidence base and illustrates why high-quality evidence from adequately

powered RCTs is needed to inform policy and practice.55 The current Cochrane review includes six RCTs,

and meta-analyses of their data suggest substantial reductions in the risk of late-onset infection and NEC

associated with lactoferrin supplementation in very preterm infants.40 However, the trials included in the

Cochrane review were small and some contained other design and methodological weaknesses that may

have introduced biases resulting in overestimation of the effect sizes.41–46 Given these concerns, the Cochrane

review authors graded the evidence for key outcomes as being of ‘low quality’ and concluded that data from

methodologically rigorous RCTs were needed to generate evidence of sufficient validity to inform policy

and practice.40

The ELFIN trial provides these data. The validity of the findings is enhanced by the quality and power of

the trial. We used best practices to limit bias, including central web-based randomisation for allocation

concealment, blinding of parents, caregivers and investigators to the group allocation, and complete

follow-up and assessment of the trial cohort with intention-to-treat analyses based on a prespecified

statistical analysis plan. The trial achieved recruitment of 2203 participants as per protocol, based on the

a priori sample size estimation. Demographic and prognostic characteristics were well-balanced between

the two groups at randomisation, with a minimisation algorithm ensuring balance for major known or

putative prognostic indicators (completed weeks of gestation, sex, single vs. multiple births) or potential

confounding influences (recruiting site). Interim analyses by the trial’s independent DMC used strict criteria

to minimise the chances of spurious findings attributable to data fluctuations before a sufficient sample size

was achieved.56,57 Adherence to the allocated interventions was high, the incidence of protocol violations

was low and outcome data were available for > 99% of the trial cohort. Event rates for the primary and

secondary outcomes were broadly similar to those that we anticipated and as have been described in other

cohort studies and RCTs involving very preterm infants.2,3 Consequently, the trial had sufficient power and

internal validity to detect reliably modest yet important effects on the risk of late-onset infection and

other morbidities.

Given the size of the ELFIN trial, with more than twice as many infants than had participated in all of the

existing trials combined, we were able to generate more precise estimates of effect size than were available

previously. The 95% CI for the relative risk estimate for the primary outcome excludes a > 14% risk reduction

and a ≥ 4% increase in risk. These estimates were consistent across completed weeks of gestation at birth,

making it unlikely that bovine lactoferrin has any important benefits for extremely preterm infants who have a

higher risk of infection. Similarly, although it is plausible that lactoferrin may have had different effects in infants

with lower levels of exposure to the immunoprotective factors present in human milk, we did not show any

interaction with the type of enteral milk feeds received during the trial period (human milk, formula or both).58

The largest previous trial, in which 331 very low birthweight infants in neonatal units in Italy participated,

showed a relative risk reduction of 66% for late-onset infection.41 Although this estimate of effect may have

been inflated by methodological weaknesses, such as the absence of predefined criteria for interim analyses,

the Italian trial differed from the ELFIN trial in other ways that could have contributed to the divergence

of findings. The participants and the intervention were broadly similar, as were enteral feeding practices,

including receipt of human breast milk versus formula milk. However, key differences in the epidemiology

of late-onset infection, as well as in infection-prevention practices and exposure to other interventions,



25

may have contributed to the difference in effects size estimates shown in the two trials. Notably, the

incidence of invasive fungal infection was very high in the Italian trial (7.7% of the control group) and a

substantial proportion of the overall effect on reducing late-onset invasive infection was due to the effect on

preventing invasive fungal infection.59 In contrast, the overall incidence of late-onset fungal infection was low

in the ELFIN trial cohort (five episodes in total), consistent with that reported in UK surveillance studies.9,60

Given that a postulated mechanism of action of lactoferrin is to reduce bowel translocation of enteric

pathogens, we assessed whether or not invasive infections with particular groups of enteric organisms

were reduced.58,61 In post hoc analyses, we did not show any evidence that lactoferrin supplementation

affected the risk of late-onset infection with different groups of infecting micro-organism including

Gram-negative bacteria (mainly Escherichia coli and other Enterobacteriaceae). This finding is consistent

with the previously largest trial, which did not show an effect of lactoferrin supplementation on the

incidence of infection with Gram-negative bacteria.41

The ELFIN trial did not show any difference in the effect of lactoferrin on the risk of late-onset infection in a

post hoc subgroup analysis of infants who had or had not received probiotic supplementation during the trial

period. A previous trial and the current Cochrane review had suggested that combining supplementation of

lactoferrin with the probiotic micro-organism Lactobacillus rhamnosus GG was associated with a greater

reduction in the risk of late-onset infection (> 70%) and NEC (> 90%) than lactoferrin alone.40,41 This raised

the possibility that the immunoprotective and prebiotic properties of lactoferrin might act synergistically

with probiotic supplementation.61 Although the ELFIN trial did not show any evidence of differential effects

depending on whether or not infants had received probiotics during the trial period, the data are not sufficient

to exclude the possibility that such prebiotic–probiotic synergism exists. A recent large cluster RCT in India has

suggested that the prophylactic administration of an oral synbiotic (prebiotic fructo-oligosaccharide combined

with probiotic Lactobacillus plantarum) reduces infection and mortality in late preterm or term newborn

infants.62 We are conducting a mechanistic study in a subgroup of ELFIN trial participants to analyse whether

or not, and how, lactoferrin supplementation affects the intestinal microbiome and metabolite profile. The

study will explore changes in microbiomic and metabolomic patterns preceding disease onset including NEC

and late-onset infection.61

Limitations

The prespecified primary outcome included ‘clinically suspected’ and ‘microbiologically confirmed’ late-

onset infection. We took this pragmatic approach because of concerns about the diagnostic accuracy of

microbiological culture of blood in this population.63 Standard microbiological culture may not detect cases

of bacteraemia or fungaemia if an insufficient volume of the infant’s blood is incubated (‘false negative’).

Conversely, microbiological cultures may also generate ‘false-positive’ results if blood sampling techniques

allow entry of contaminating micro-organisms (typically from the infant’s skin). To mitigate these potential

sources of bias, we used an established consensus case definition that (1) required additional evidence of

infection (clinical signs or biomarkers) and (2) mandated that clinicians indicate an intention to treat the

infant with antibiotics or antifungals for at least 5 days.2,3

Typically, microbiological confirmation was obtained by culture of potentially pathogenic bacteria or

fungi from an infant’s blood or CSF sample, or from another normally sterile tissue space. The outcome

definition included infection with coagulase-negative Staphylococci, provided that these were not a mixed

flora but excluded micro-organisms that were likely to be skin contaminants (diphtheroids, micrococci or

propionibacteria). This approach is consistent with standard clinical practice and surveillance protocols in

the UK and elsewhere. The case definition of late-onset infection did not include urinary tract infection or

radiologically confirmed pneumonia, as these are not accurate and reliable in very preterm infants in the

absence of bacteraemia.64

DISCUSSION AND CONCLUSIONS


26

Secondary outcomesEstimates for the secondary outcomes indicated consistently that lactoferrin supplementation does not have

important effects on the risk of major morbidities. We prespecified an analysis of the effect on a composite

of infection, NEC, BPD, ROP and mortality. The adjusted RR point estimate for this secondary outcome

was 1.01, with a 99% CI excluding a > 6% reduction and a ≥ 8% increase in risk. We plan to increase

the precision of these estimates of effect on rarer secondary outcomes by combining these data in a

meta-analysis with other trials, including a recently completed Australasia RCT (n = 1500) of bovine

lactoferrin supplementation for very low birthweight infants (Lactoferrin Infant Feeding Trial; see

www.anzctr.org.au/ACTRN12611000247976.aspx).65

Cost analysesAs late-onset infection and NEC are the major reasons for receipt of invasive interventions and higher levels

of ‘categories of care’ in very preterm infants, it is not surprising that we did not show any effects on the level

of exposure to antimicrobial agents or on the duration of hospitalisation or stay in intensive or special care

settings. Given that the ELFIN trial did not show any differences between groups in the risk of morbidity or

on levels of care received, we did not undertake detailed analyses of health-care costs as had been proposed

in our approved funding application and trial protocol. We did not conduct a within-trial health economic

analysis or use these data in a model to explore long-term family and health service costs, as these are driven

mainly by the consequences of infection and other morbidity during the initial hospitalisation. Without

evidence of clinical effectiveness on these infant-important outcomes, we considered a cost-effectiveness

analysis of lactoferrin supplementation to be futile.66

Qualitative analysis and parent viewsA qualitative analysis and exploration of participants’ parents views and expectations has been undertaken

in collaboration with the SIFT investigators.48 Given that this study included SIFT participants predominantly

(with few ELFIN trial participants), the findings will be reported within the SIFT report.

Long-term outcomesWe do not plan to apply for permission and additional funding to assess longer-term outcomes of trial

participants. We specified in our funding application and protocol that if the trial did not detect statistically

significant or clinically important differences in the in-hospital outcomes then follow-up will not be undertaken

because any between-group differences in growth and neurodevelopmental outcomes are predicated largely

on differences in the incidence of late-onset infections, NEC and associated morbidities.5,6,11 As these were not

shown, there is no longer an impelling rationale for expecting lactoferrin supplementation to have an impact

on long-term growth or development.

Applicability

The ELFIN trial findings are likely to be applicable in the UK and internationally. Participants were enrolled

in 37 neonatal units across the country, providing broad geographical, social and ethnic representation.

Many infants who were enrolled in a recruiting site were transferred subsequently to another neonatal unit,

which was typically closer to the family home, for ongoing care. Trial participation continued in another

97 neonatal units and this practice mirrors managed clinical network care pathways for very preterm infants

in the UK.

The trial population was representative of very preterm infants cared for within health-care facilities in

well-resourced health services and included a substantial proportion of extremely preterm infants (37%)

and infants with other putative risk factors for neonatal morbidity, such as prolonged rupture of maternal

amniotic membranes (25%) and evidence of absent or reverse end diastolic flow in the umbilical artery

(12%). Overall, about 30% of participants acquired a microbiologically confirmed or clinically suspected

late-onset infection, and about 17% in total had a microbiologically confirmed infection, consistent with

rates reported from cohort studies and other RCTs. Similarly, the incidence of NEC (about 5%) was similar

to rates reported in large, population-based surveillance and cohort studies and RCTs.67



27

https://www.anzctr.org.au/ACTRN12611000247976.aspx

Implications for practice

The ELFIN trial does not support the routine use of enteral bovine lactoferrin supplementation to prevent

late-onset infection or other morbidity or mortality in very preterm infants.

Implications for research

Research efforts should continue to investigate the aetiology, epidemiology and pathogenesis of late-onset

infection and related morbidities, and to develop, refine and assess other interventions that may prevent or

reduce adverse acute and long-term consequences for very preterm infants and their families.

DISCUSSION AND CONCLUSIONS


28

Acknowledgements

We are grateful to all of the parents of participating infants and to all staff and carers in recruiting and

continuing care sites. We thank the members of the independent DMC and TSC, Yan Hunter-Blair

and colleagues at the Newcastle Specials Pharmacy team, Tatua Dairy Co-operative, New Zealand, and the

administrative and support colleagues at the NPEU CTU.

Independent Trial Steering Committee

Richard Cooke (chairperson), Fan Hutchison (deputy chairperson), Andrew Ewer, Jennifer Hellier and

Paul Mannix.

Independent Data Monitoring Committee

Henry Halliday (chairperson), Nim Subhedar, Michael Millar, Alison Baum and Mike Bradburn.

Ethics approval

National Research Ethics Service Committee East Midlands – Nottingham 2 (reference number 13/EM/0118,

02/04/2013).

Contributions of authors

James Griffiths (Trial Manager), Paula Jenkins (Trial Research Nurse), Monika Vargova (Administrator

and Data Co-ordinator), Ursula Bowler (Senior Trials Manager), Andrew King (Head of Trials

Programming), David Murray (Senior Trials Programmer), Paul T Heath (Co-investigator, chairperson of

the blinded end-point review committee) and William McGuire (Chief Investigator) were responsible for

the data collection and management.

Edmund Juszczak (NPEU CTU Director), Janet Berrington (Co-investigator), Nicholas Embleton

(Co-investigator), Jon Dorling (Co-investigator), Paul T Heath, William McGuire and Sam Oddie

(Co-investigator) were responsible for the study design.

Louise Linsell (Trial Statistician), Christopher Partlett (Trial Statistician), Edmund Juszczak,

Paul T Heath and William McGuire were responsible for the data analysis.

Mehali Patel (Patient and Public Involvement Representative), Edmund Juszczak, Janet Berrington,

Nicholas Embleton, Paul T Heath, William McGuire and Sam Oddie were responsible for the data

interpretation.

James Griffiths, Edmund Juszczak, Louise Linsell, Christopher Partlett and William McGuire were

responsible for the report writing.

All authors approved the final draft of the manuscript.



29

Publications

ELFIN Trial Investigators Group. Lactoferrin immunoprophylaxis for very preterm infants. Arch Dis Child

Fetal Neonatal Ed 2013;98:F2–4.

The ELFIN Trial Investigators Group. Enteral lactoferrin supplementation for very preterm infants:

a randomised controlled trial. Lancet 2018; in press.

Data-sharing statement

All data requests should be submitted to the corresponding author for consideration. Please note that

exclusive use will be retained until the publication of major outputs. Access to anonymised data may be

granted following review.

ACKNOWLEDGEMENTS


30

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https://doi.org/10.1002/hec.1093




Appendix 1 Recruiting neonatal units

Recruiting sites PI Research nurse

Altnagelvin Area Hospital Mary Ledwidge Julie Brown

Birmingham Heartlands Hospital Imogen Story Natalie Albrighton

Birmingham Women’s Hospital Gemma Holder Rachel Jackson/Elizabeth Simcox/Heather Barrow

Bradford Royal Infirmary Sam Oddie Kelly Young/Trudy Booth

Calderdale Royal Hospital Pamela Ohadike Salamiah Burgess

University Hospital Coventry Sarah Ellis Kerri McGowan/Nicola Watts

Derriford Hospital, Plymouth Rima Vaikute Sarah-Jane Sharman

Great Western Hospital, Swindon Girish Gowda Rebecca Elliott-Jones

Hull Royal Infirmary Helen Yates Leanne Sherris

James Cook University Hospital, Middlesbrough Shalabh Garg Amanda Forster/Helena Smith

Jessop Wing – Sheffield Teaching Hospital Liz Pilling Pauline Bayliss

John Radcliffe Hospital, Oxford Charles Roehr Sheula Barlow/Sharon Baugh

Leeds General Infirmary Kathryn Johnson Suzanne Laing

Leicester Royal Infirmary Elaine Boyle Marie Hubbard/Rosalind Astles

Norfolk and Norwich University Hospital Paul Clarke Karen Few

Nottingham City Hospital Dushyant Batra Yvonne Hooton/Helen Navarra

Pinderfields Hospital, Wakefield David Gibson Gail Castle

Princess Anne Hospital, Southampton Mark Johnson Jenny Pond/Philippa Crowley/Jane Rhodes-Kitson

Princess Royal Maternity Hospital, Glasgow Helen Mactier Isobel Crawford

Queen Alexandra Hospital, Portsmouth Tim Scorrer Michelle Pople/Michele Voysey

Nottingham University Hospital Jon Dorling Yvonne Hooton/Helen Navarra

Royal Cornwall Hospital, Truro Yadlapalli Kumar Barbara Bromage

Royal Devon and Exeter NHS Foundation Trust David Bartle Jacqui Tipper/Jenny Cunningham

Royal Hospital for Children, Glasgow Colin Peters Lorna McKay

Royal Infirmary of Edinburgh David Quine Lynn Clark

Royal Maternity Hospital, Belfast Stanley Craig Muriel Millar

Royal Preston Hospital Richa Gupta Claire Lodge

Royal Victoria Infirmary, Newcastle upon Tyne Nick Embleton Julie Groombridge

Singleton Hospital, Swansea Jean Matthes Amanda Cook

St George’s Hospital, London Nigel Kennea Vana Wardley/Naomi Hayward

St Peter’s Hospital, Chertsey Peter Reynolds Nicky Holland

Sunderland Royal Hospital Ruppa Geethanath Natalie Talbot

University Hospital of North Tees Sundaram Janakiraman Alex Ramshaw

Victoria Hospital, Kirkcaldy Sean Ainsworth Debbie Johnston

William Harvey Hospital, Ashford Vimal Vasu Shermi George



37

Recruiting sites PI Research nurse

Wishaw General Hospital CM Manjunatha Denise Vigni

York District Hospital William McGuire Anna Clayton

APPENDIX 1


38

Appendix 2 Continuing care sites

Accrington Victoria Hospital; Airedale General Hospital; Antrim Area Hospital; Barnsley Hospital; Basildon

University Hospital; Basingstoke and North Hampshire Hospital; Bassetlaw Hospital; Birmingham

Children’s Hospital; Birmingham City Hospital; Borders General Hospital; Broomfield Hospital; Burnley

General Hospital; Chesterfield Royal Hospital; Chorley and South Ribble Hospital; Colchester General

Hospital; County Hospital, Stafford; Craigavon Area Hospital; Crosshouse University Hospital; Croydon

University Hospital; Darlington Memorial Hospital; Diana Princess of Wales Hospital, Grimsby; Doncaster

Royal Infirmary; Dorset County Hospital; Dumfries and Galloway Royal Infirmary; Forth Valley Royal

Hospital; George Eliot Hospital, Nuneaton; Glangwili General Hospital; Gloucestershire Royal Hospital;

Good Hope Hospital, Sutton Coldfield; Great Ormond Street Hospital for Children, London; Harrogate District

Hospital; Horton General Hospital; Ipswich Hospital; James Paget University Hospital, Great Yarmouth;

King’s Mill Hospital, Sutton-in-Ashfield; Lincoln County Hospital; Liverpool Women’s Hospital; Maidstone

Hospital; Medway Maritime Hospital, Gillingham; Milton Keynes General Hospital; Musgrove Park Hospital,

Taunton; Northampton General Hospital; North Manchester General Hospital; Northumbria Specialist

Emergency Care Hospital, Cramlington; Pilgrim Hospital, Boston; Poole Hospital; Princess of Wales Hospital,

Bridgend; Queen Elizabeth Hospital, Gateshead; Queen Elizabeth Hospital, King’s Lynn; Queen Elizabeth

The Queen Mother Hospital, Margate; Queen’s Hospital, Burton on Trent; Queen’s Hospital, Romford;

Raigmore Hospital, Inverness; Rotherham General Hospital; Royal Berkshire Hospital, Reading; Royal

Blackburn Hospital; Royal Bolton Hospital; Royal Derby Hospital; Royal Hampshire County Hospital,

Winchester; Royal Oldham Hospital; Royal Shrewsbury Hospital; Royal Surrey County Hospital, Guildford;

Russells Hall Hospital, Dudley; Salisbury District Hospital; Scarborough General Hospital; Scunthorpe

General Hospital; Southend Hospital, Westcliff-on-Sea; Southport Hospital; South Tyneside District Hospital,

South Shields; Stepping Hill Hospital, Stockport; St John’s Hospital, Livingstone; St Mary’s Hospital,

Newport; Stoke Mandeville Hospital, Aylesbury; St Richard’s Hospital, Chichester; The County Hospital,

Hereford; The Cumberland Infirmary, Carlisle; Torbay Hospital, Torquay; Tunbridge Wells Hospital; Ulster

Hospital, Dundonald; University Hospital Lewisham; University Hospital of North Durham, Durham;

Warwick Hospital; Watford General Hospital; West Cumberland Hospital, Whitehaven; West Suffolk

Hospital, Bury St Edmunds; Withybush General Hospital, Haverfordwest; Worcestershire Royal Hospital,

Worcester; Worthing Hospital; and Wrightington Hospital, Wigan.



39

Appendix 3 Preparation of InvestigationalMedicinal Product for administration

1. Verify that the pack ID number on the pharmacy pot matches the pack ID allocated to the infant

(stated on the randomisation confirmation e-mail and to be recorded on the daily dosing log).

2. Add 4 ml of sterile water (supplied in plastic vial) plus 1 ml of either expressed breast milk or formula

(if expressed breast milk is not available) to the pharmacy pot, which contains 375 mg of either

lactoferrin or sucrose placebo.

3. Seal the pot with the lid and shake vigorously by hand for 30 seconds.

4. Leave the pot at room temperature for 30 minutes.

5. Using a syringe, draw off suspension (2 ml/kg body weight up to a maximum of 4 ml) for nasogastric/

orogastric or oral administration (via spoon/cup/syringe or bottle). [Participating centres were supplied

with oral syringes if their standard oral syringe was not compatible with the lactoferrin/placebo

pot insert].

6. Trial IMP normally to be given once daily. For very small infants, clinicians or caregivers may choose to

administer the daily dose in two aliquots. If these are to be given > 30 minutes apart, then a fresh dose

should be prepared as above for each.

7. If there was any concern about acute intestinal inflammation or perforation then the dose could be

omitted. Whether or not doses were omitted at other times when the infant was unwell or demonstrated

enteral feeds intolerance was at the discretion of the attending consultant paediatrician.



41

Appendix 4 Case definition of necrotisingenterocolitis

Necrotising enterocolitis may be diagnosed at surgery, at post-mortem examination or clinically and

radiologically using the following criteria.

At least one of the following clinical signs present:

l bilious gastric aspirate or emesisl abdominal distensionl occult or gross blood in stool (no fissure).

In addition, at least one of the following radiological features present:

l pneumatosis intestinalisl hepatobiliary gasl pneumoperitoneum.

Infants who satisfy the definition of NEC above but are found at surgery or post-mortem examination for

that episode to have a ‘focal gastrointestinal perforation’ should be coded as having ‘focal gastrointestinal

perforation’, not as having NEC.



43

Appendix 5 British Association of PerinatalMedicine: ‘categories of care’

URL: www.bapm.org/sites/default/files/files/CatsofcarereportAug11.pdf (accessed 29 June 2018).

Intensive care

General principle: this is care provided for infants who are the most unwell or unstable and have the

greatest needs in relation to staff skills and staff-to-patient ratios.

Definition of intensive care dayAny day when an infant receives any form of mechanical respiratory support via a tracheal tube.

Both non-invasive ventilation [e.g. nasal continuous positive airways pressure (CPAP)] and parenteral nutrition.

l Day of surgery (including laser therapy for ROP).l Day of death.l Any day receiving any of the following:

¢ presence of an umbilical arterial line¢ presence of an umbilical venous line¢ presence of a peripheral arterial line¢ insulin infusion¢ presence of a chest drain¢ exchange transfusion¢ therapeutic hypothermia¢ prostaglandin infusion¢ presence of replogle tube¢ presence of epidural catheter¢ presence of silo for gastroschisis¢ presence of external ventricular drain¢ dialysis (any type).

High-dependency care

General principle: this is care provided for infants who require highly skilled staff but where the ratio of

nurses to patients is less than that in intensive care.

Definition of high-dependency care dayAny day when an infant does not fulfil the criteria for intensive care where any of the following apply:

Any day when an infant receives any form of non invasive respiratory support (e.g. nasal CPAP).



45

https://www.bapm.org/sites/default/files/files/CatsofcarereportAug11.pdf

Any day receiving any of the following:

parenteral nutrition

continuous infusion of drugs (except prostaglandin and/or insulin)

presence of a central venous or long line (peripherally inserted central catheter)

presence of a tracheostomy

presence of a urethral or suprapubic catheter

presence of transanastomotic tube following oesophageal atresia repair

presence of nasopharyngeal airway/nasal stent

observation of seizures or cerebral function monitoring

barrier nursing

ventricular tap.

Special care

General principle: special care is provided for infants who require additional care delivered by the neonatal

service but do not require either intensive or high-dependency care.

Definition of special care dayAny day where an infant does not fulfil the criteria for intensive or high-dependency care and requires any

of the following:

oxygen by nasal cannula

feeding by nasogastric, jejunal tube or gastrostomy

continuous physiological monitoring (excluding apnoea monitors only)

care of a stoma

presence of intravenous cannula

phototherapy

special observation of physiological variables at least 4 hourly.

APPENDIX 5


46

Appendix 6 Data collection forms

URL: www.npeu.ox.ac.uk/elfin/data-collection-forms (accessed 24 July 2018).

Form Purpose

Trial entry form The entry form contains sections to be completed before, during and after randomisation,and collects the infant’s baseline characteristics

Daily dosing log To be completed daily during the treatment period (once the infant receives milk feeds of12 ml/kg/day until 34 weeks’ postmenstrual age) to document the administration oflactoferrin or placebo, type of milk given and use of antibiotic and antifungal drugs

Late-onset infection form To report each episode of microbiologically confirmed or clinically suspected late-onsetinvasive infection

Gut signs form To report each episode whenever an infant has received ≥ 5 days of treatment for gut signs,if they are transferred with gut signs, or if they have died from gut signs

Hospital transfer anddischarge form

To be completed by each recruiting, continuing care or data collection site whenever aparticipating infant is discharged home, is transferred to another unit, or has died

Discontinuation ofintervention

To be completed if lactoferrin or placebo is permanently discontinued early (by clinician orparental decision) or where parents choose to withdraw their infant from the trial

SAE/SUSAR form Should be completed for all SAEs that are ‘unexpected’ and sent to the NPEU CTU within24 hours of becoming aware of the event

Incident form To report any deviation from the protocol, trial-specific procedures or good clinical practice



47

https://www.npeu.ox.ac.uk/elfin/data-collection-forms

Appendix 7 Safety reporting: definitions

Adverse event

Any untoward medical occurrence in a patient or clinical investigation participant who has been administered

a medicinal product, which does not necessarily have to have a causal relationship with this treatment

(the study medication).

An adverse event can therefore be any unfavourable and unintended sign (including an abnormal laboratory

finding), symptom or disease temporally associated with the use of the study medication, whether or not it

is considered to be related to the study medication.

Adverse reaction

All untoward and unintended responses to a medicinal product related to any dose.

The phrase ‘responses to a medicinal product’ means that a causal relationship between a study medication

and an adverse event is at least a reasonable possibility (i.e. the relationship cannot be ruled out). All cases

judged by either the reporting medically qualified professional or the sponsor as having a reasonable

suspected causal relationship to the study medication qualify as adverse reactions.

Serious adverse event

Adverse events are defined as serious if they:

l result in deathl are life-threateningl require inpatient hospitalisation or prolongation of existing hospitalisationl result in persistent or significant disability/incapacityl are a congenital anomaly/birth defectl are other important medical events.

Note that other events that may not result in death, are not life-threatening or do not require hospitalisation

may be considered SAEs when, based on medical judgement, the event may jeopardise the patient

and may require medical or surgical intervention to prevent one of the outcomes listed above. The term

‘life-threatening’ refers to an event in which the patient was at risk of death at the time of the event; it does

not refer to an event that hypothetically might have caused death if it were more severe.

Serious adverse reaction

A serious adverse reaction (SAR) is a SAE that is considered to have been caused by the administration of

the trial medication. For a SAE to be considered a SAR, there must be a reasonable probability that it was

related to the administration of the IMP.



49

Suspected unexpected serious adverse reaction

This is a SAR, the nature or severity of which is not consistent with the known safety profile of the trial

medication (e.g. investigator’s brochure for an unapproved investigational product or summary of product

characteristics for an approved product).

Foreseeable (‘expected’) serious adverse events

The following are SAEs that could be reasonably expected to occur in this population of infants during the

course of the trial or form part of the outcome data. They do not require reporting by the trial centres

as SAEs:

l death (unless unexpected in this population)l NEC or focal intestinal perforationl BPD or chronic lung diseasel intracranial abnormality (haemorrhage or focal white matter damage) on cranial ultrasound scan or

other imagingl pulmonary haemorrhagel patent ductus arteriosusl ROP.

APPENDIX 7


50

Appendix 8 Summary of changes to thestudy protocol

P rotocol is available at www.npeu.ox.ac.uk/elfin/protocols (accessed 24 July 2018) and in the

Neonatology article.47



51

http://www.npeu.ox.ac.uk/elfin/protocols

Amendment

Date of RECfavourableopinion

Date ofMHRA approval Document Description

Amend 1 14 October 2013 –

(Prior to ClinicalTrials Authorisationapplication)

Protocol version 2 l The procedure for making up the intervention was changed to reduce the total fluid volumeto 5ml (1 ml milk + 4ml water). The concentration of the solution to be administered(75 mg/ml) and dose (150mg/kg/day) is unchanged from the original application

l The exclusion criteria were clarified. One serious congenital anomaly is sufficient forexclusion (changed from anomalies)

l Clarification was added that it is acceptable to recruit infants to both the ELFIN trial and SIFTl The definitions of microbiologically confirmed and clinically suspected late-onset infection

were edited for clarityl ‘Multiple births’ was added as a minimisation factor at randomisationl Text was added to explain that participants will be ‘flagged’ by the Health and Social Care

Information Centrel Changes to appendix 3: IMP management explaining the procedure for making up

the interventionl The projected recruitment rate in appendix 4 was revised slightly upwards

IMP dossier version 2 l Ampoules of sterile water will be sourced from clinical areas rather than being supplied withthe IMP

l Owing to total fluid volume per dose being reduced from 6ml to 5 ml, the amount oflactoferrin in each individual dose was reduced to 375 mg so that the resulting solutionremained 75mg/ml as in the original application

l Inclusion of a Press-In Bottle Adapter in each container of IMP, compatible with oral syringesl Updated the certificates and compliance statements included as appendices to the most

recent available versions

Consent form version 2 l Sections were added for the name of the participant from whom and hospital at whichconsent was taken

PIL version 2 l Added the statement ‘We will keep your name, address and other contact details. TheHealth and Social Care Information Centre and other central UK NHS bodies will be used tokeep in touch with you and provide information about your baby’s health status’

l Added the statement ‘Unidentifiable data from this study may be shared with other groupswho are carrying out similar work’ to cover anonymised data-sharing after the trial is over,in line with the requirements of the funder

l Removed the word ‘independent’ in relation to the charity Bliss

ELFIN statement ofresponsibilities version v1

l This document was introduced to describe the arrangements for recruiting, continuing careand data collection sites

APPENDIX

8

NIHRJournalsLib

rary

www.jo

urnalslib

rary.n

ihr.a

c.uk

52

Amendment



Amend 2 3 January 2014 17 January 2014 IMP dossier version 3 l A provision for the use of non-automated filling of doses for up to 82 subjects for the pilotphase of the trial. This is necessary because the auger filler to be used for automated fillingwill not be installed and commissioned in time for the proposed start of recruitment

l A change to the HPLC assay for bovine lactoferrin

Amend 3 27 May 2014 – ELFIN/SIFT summaryleaflet version 1

l To add a joint summary leaflet to introduce both the ELFIN trial and SIFT to parents, whererecruitment to both trials is being considered (SIFT submitted an identical amendment to theethics committee)

Amend 4 11 July 2014 21 July 2014 – l Notification of temporary halt to trial. The circumstances leading to the temporary halt aredescribed fully in the amendment 4 covering letter

Amend 5 13 October 2014 3 November 2014 IMP dossier version 4 l Drug substance will be flushed with nitrogen when added to the hopper of the servo augerfiller machine

l Text for manual filling was amended to clarify that this process was used for batchesIMPNS4B002 and IMPNS4C001. Further batches will be manufactured by automatedprocess (using auger servo filler machine)

l Six sealed containers will be packed in a nitrogen-flushed, labelled aluminium pouch, linedwith polyethyltoluene and low-density polyethylene

l Four pouches of six containers (24 containers total) will be packed into one labelledcardboard outer

l Based on the data presented in Table 2, maximum expiry will be limited to 2 years fromdate of manufacture, assuming storage at ≤ 30 °C

l The stability testing programme will be conducted at 30 °C and 40 °C, in line with therevised storage requirements of the product

GMP label version 2 l Storage requirements have been amended to ‘Store at or below 30 °C’. The original labelspecified to store at or below 25 °C

Amend 6 29 October 2014 – NICU parent posterversion 1, antenatal wardposter version 1

l Poster intended for viewing by pregnant women and/or their partners in antenatal areasl Poster intended for viewing by parents in the neonatal unit

Amend 7 23 April 2015 – – l Addition of further recruiting sites in England

Amend 8 31 July 2015 – – l Addition of recruiting sites in England, Northern Ireland and Scotland

PIL version 3 l Added England/Northern Ireland/Scotland specific variants of version 3l Updated contact details for the charity Blissl N.B. REC considered these changes non-substantial

Consent form version 3 l References PIL version 3 (Considered by REC to be non-substantial)

DOI:10.3310/hta22740

HEALTHTECHNOLOGYASSESSMENT2018

VOL.22

NO.74

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53

Amendment



Amend 9 10 September 2015 – – l Conversion of some continuing care sites in England to recruiting sites

Amend 10 – 16 December 2015 – l Removed requirement for temperature monitoring of IMP at continuing care sites

Amend 11 18 December 2015 31 December 2015 Protocol version 2.1 l Single change to protocol to remove requirement for research nurse reports to projectmanagement group (not implemented as included in protocol version 3.0)

Amend 12 21 January 2016 – – l Conversion of additional continuing care sites in England to recruiting sites

Amend 13 8 March 2016 4 March 2016 Protocol version 3.0 l Section 4.7 describing existing RCTs of lactoferrin supplementation in preterm babies wasupdated to reflect the recent 2015 Cochrane review68

l Section 7.2 was expanded to clarify that appropriately qualified and experienced neonatalnurses may be delegated by the PI to assess eligibility

l Sections 7.8.1 and 7.8.2 were changed to clarify how doses of IMP should be calculatedagainst an infant’s current working weight and to better emphasise the pragmatic natureof this trial

l Section 7.8.3 was added to clarify that independent nurse prescribers may be delegated bythe PI to prescribe IMP, provided this is consistent with local trust policy

l Section 7.10 was changed to reflect an updated procedure for unblinding a participant inthe event of an emergency

l Section 7.16 was changed so that the definition of end of trial is now the date at which thetrial database is locked

l Section 9.2 was added clarify the reference safety information to be used for the assessmentof adverse drug reactions

l Section 9.3.1 was changed to clarify that adverse events not meeting the criteria forseriousness will not be collected for this trial

Amend 14 9 August 2016 – – l Added additional recruiting sitesl Change of some PIs at existing sites

Amend 15 22 November 2016 – – l Added a Wales-specific variant of ELFIN PIL version 3l Added continuing care sites in Walesl Change of some PIs at existing sites

Amend 16 6 September 2017 – – l Change of a PI at an existing site

APPENDIX

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54

Amendment



Amend 17 15 March 2018 15 March 2018 Protocol version 4.0 l The addition of a secondary outcome: microbiologically confirmed late-onset invasive infectionl Two secondary outcomes have been combined: total number of days of administration of

antibiotics administered per infant from 72 hours until death or discharge from hospital,and total number of days of administration of antifungal agents per infant. The revisedsecondary outcome is as follows: total number of days of administration of antibiotics orantifungals (excluding prophylactic doses) from the commencement of dosing with the IMPuntil 34 weeks’ postmenstrual age. The revised outcome more closely corresponds to theperiod when the IMP bovine lactoferrin is administered

l An addition to the protocol to clarify that, if a participant is not discharged to home within6 months (26 weeks) of the date of birth, data collection for that participant will becompleted at 6 months (26 weeks) from the date of birth

l Recruitment tables and graphs in appendix 4 were updated to reflect the actual recruitmentand date that recruitment to the trial ended

Amend 18 Pending Pending See protocol version 5.0for track changes

l Submitted 27 June 2018l Declaration NOT to conduct previously specified health economics analysis

HPLC, high-performance liquid chromatography; NICU, neonatal intensive care unit.

DOI:10.3310/hta22740

HEALTHTECHNOLOGYASSESSMENT2018

VOL.22

NO.74

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55

Appendix 9 Withdrawals from intervention byrandomisation group

Reason

Trial group, n

Lactoferrin (n= 1099)a Placebo (n= 1104)a

Clinical decision 4 1

Consent remains (4) (1)

Consent completely withdrawn (0) (0)

Parental wish 15 14

Consent remains (15) (11)

Consent completely withdrawn (0) (3)

Otherb 1 0

Total 20 15

a Includes all infants randomised.b Baby transferred to a hospital that refused to accept or administer the study intervention.



57

Appendix 10 Group allocation per recruiting site

Centre

Trial group, n (%)


Jessop Wing, Sheffield 24 (2.2) 24 (2.2)

Royal Infirmary of Edinburgh 25 (2.3) 26 (2.4)

Princess Royal Maternity Hospital, Glasgow 26 (2.4) 27 (2.5)

Wishaw General Hospital 20 (1.8) 23 (2.1)

Royal Maternity Hospital, Belfast 20 (1.8) 20 (1.8)

James Cook University Hospital 76 (6.9) 70 (6.4)

Nottingham City Hospital 21 (1.9) 19 (1.7)

Queen’s Medical Centre, Nottingham 15 (1.4) 15 (1.4)

Birmingham Heartlands Hospital 16 (1.5) 14 (1.3)

Birmingham Women’s Hospital 52 (4.7) 54 (4.9)

Sunderland Royal Hospital 21 (1.9) 25 (2.3)

Altnagelvin Area Hospital, Londonderry 5 (0.5) 5 (0.5)

University Hospital Coventry 33 (3.0) 35 (3.2)

Royal Victoria Infirmary, Newcastle 68 (6.2) 66 (6.0)

University Hospital of North Tees 32 (2.9) 32 (2.9)

John Radcliffe Hospital, Oxford 15 (1.4) 17 (1.5)

Hull Royal Infirmary 17 (1.5) 17 (1.5)

Bradford Royal Infirmary 109 (9.9) 109 (9.9)

Calderdale Royal Hospital 10 (0.9) 10 (0.9)

Derriford Hospital, Plymouth 4 (0.4) 4 (0.4)

Great Western Hospital, Swindon 3 (0.3) 3 (0.3)

Leeds General Infirmary 83 (7.6) 82 (7.4)

Leicester Royal Infirmary 52 (4.7) 53 (4.8)

Norfolk and Norwich University Hospital 33 (3.0) 30 (2.7)

Pinderfields General Hospital, Wakefield 6 (0.5) 4 (0.4)

Princess Anne Hospital, Southampton 28 (2.6) 32 (2.9)

Queen Alexandra Hospital, Portsmouth 83 (7.6) 86 (7.8)

Royal Cornwall Hospital, Truro 15 (1.4) 12 (1.1)

Royal Devon and Exeter Hospital 13 (1.2) 9 (0.8)

Royal Hospital for Children, Glasgow 18 (1.6) 20 (1.8)

Royal Preston Hospital 16 (1.5) 15 (1.4)

Singleton Hospital, Swansea 14 (1.3) 12 (1.1)

St George’s Hospital, London 30 (2.7) 32 (2.9)

St Peter’s Hospital, Chertsey 32 (2.9) 32 (2.9)

William Harvey Hospital, Ashford 26 (2.4) 29 (2.6)

York Hospital 15 (1.4) 15 (1.4)

Victoria Hospital, Kirkcaldy 22 (2.0) 23 (2.1)



59

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This report presents independent research funded by the

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